Dynamic Expression Pattern of SERPINA1 Gene from Duck ...downloads.hindawi.com/journals/bmri/2019/1321287.pdf · Dynamic Expression Pattern of SERPINA1 Gene from Duck (Anas platyrhynchos)

Research ArticleDynamic Expression Pattern of SERPINA1 Gene from Duck(Anas platyrhynchos)

Tiantian Gu1 NingzhaoWu1 Yang Zhang1 Yu Huang2 Jinping Du3 Lizhi Lu4

Xinsheng Wu1 Qi Xu 1 and Guohong Chen 1

1Key Laboratory of Animal Genetics amp Breeding and Molecular Design of Jiangsu Province Yangzhou University Yangzhou225009 China

2Institute of Animal Science Fujian Academy of Agricultural Sciences Fujian 350000 China3Institute of Animal Science Hubei Academy of Agricultural Sciences Wuhan 430000 China4Institute of Animal Science Zhejiang Academy of Agricultural Sciences Hanghzou 310000 China

Correspondence should be addressed to Qi Xu xuqiyzueducn and Guohong Chen ghchenyzueducn

Received 9 October 2018 Revised 30 January 2019 Accepted 3 March 2019 Published 19 March 2019

Academic Editor Kui Li

Copyright copy 2019 Tiantian Gu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

SERPINA1 is a member of serine protease inhibitors and is increasingly considered to be a regulator of innate immunity in humanand animals However the expression and function of SERPINA1 gene in immune defense against viral infection remain unknownin ducks The full-length du SERPINA1 cDNA sequence was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) It contained 1457 nucleotide including 47-bp 5rsquo UTR 135-bp 3rsquo UTR and1275-bp open reading frame (ORF) and encodes a 424-amino acid proteinThen the tissue expression profile of du SERPINA1 genewas determined Real-time quantitative polymerase chain reaction (real-time qPCR) analysis revealed that du SERPINA1mRNA isubiquitous in various tissues but higher expression levelswere observed in lung and liver tissues In addition the expression patternwas investigated when the ducklings were challenged with duck hepatitis virus 1(DHV-1) and polyriboinosinic polyribocytidylicacid (poly IC) After DHV-1 injection or poly IC treatment du SERPINA1mRNAwas up-regulated in the liver and kidney tissuesHowever the peak time in two tissues was not consistent In kidney the expression lever of SERPINA1 increased immediately afterthe treatment while in liver tissue it kept steady until 12 h post-infection Our results indicate that SERPINA1 has an active role inthe antiviral response and thus improve our understanding of the role of this protein

1 Introduction

Serpins as a superfamily of serine protease inhibitors play avital role in complement regulation inflammation angiogen-esis tumor suppression apoptosis and other physiologicalprocesses [1 2]There is ample clinical evidence thatmutationin this gene could cause emphysema or liver disease whichshowed a serious impact on the function and homeostasisof tissues and organs [3] So far sixteen clades have beenidentified designated A through P with an additional 10serpins that are unclassified ldquoorphansrdquo [1] SERPINA1 alsoknown as Alpha-1 anti-trypsin (AAT) is a vital member ofthe SERPIN superfamily which plays an important role in

anti-inflammatory properties [4ndash6] Recent findings indicatethat Alpha1-antitrypsin may not only prevent damage fromproteolysis but may also specifically degrade elastin in tissuesand organs and inhibit some immune pathways to affectregulation of innate immunity [4ndash6]

Ducks as a feasible model play an important role instudying avian influenza and human hepatitis and hasraised interest in the duck immune system [7] DHV-1 isa small RNA virus causing high mortality in ducks (Anasplatyrhynchos) especially in younger ducklings In order toreduce the effect of DHV-1 a large number of studies havebeen reported on this virus [8ndash14] In our previous studywe identified differentially expressed sequence tags (ESTs)

HindawiBioMed Research InternationalVolume 2019 Article ID 1321287 8 pageshttpsdoiorg10115520191321287

2 BioMed Research International

Table 1 Primers used in the study

Primer name Primer sequence (5rsquo997888rarr3rsquo) Annealing temperature (∘C) Applicationdu SERPINA1-F ATGAAAATGAAGTCTGCACTG 55 RT-PCRdu SERPINA1-R CTAGCCTACCTTGAGAGTTGGGdu SERPINA1 5rsquo Router GTTCTTGTTACCTTCCTCCT 60 5rsquo RACE-PCRdu SERPINA1 5rsquo Inner GGAGAGGCTCAAAGGAdu SERPINA1 3rsquo Router AGGTGACCGCCTTATGGTTTCC 60 3rsquo RACE-PCRdu SERPINA1 3rsquo Inner AATAATGCTGACCTCTCTGGAGTGGCqdu SERPINA1-F AATACTAGCCAGCAAAACGAA 60 RT-qPCRqdu SERPINA1-R TGAGCCAAGTTTTCACCTTCGAPDH-F TGCTAAGCGTGTCATCATCT 60 RT-qPCRGAPDH-R AGTGGTCATAAGACCCTCCA

of SERPINA1 using a suppression subtractive hybridization(SSH) cDNA library of 3-day-old ducklings treated withDHV-1 [15] Our study showed that during DHV-1 and polyIC infection the expression of SERPINA1 mRNA was up-regulated In this study we aim to expand those preliminaryresults by assessing tissue-specific gene expression and thedynamic expression change of the SERPINA1 gene againstthe virus and thereby provide a theoretical basis for futureimmune pathological studies

2 Materials and Methods

21 Ethics Approval and Consent to Participate The animalexperiment was reviewed and approved by the InstitutionalAnimal Care and Use Committee of Yangzhou University(approval number 151-2014) Procedures were performedin accordance with the Regulations for the Administrationof Affairs Concerning Experimental Animals (YangzhouUniversity China 2012) and the Standards for the Adminis-tration of Experimental Practices (Jiangsu China 2008) Wealso confirm that all efforts were made to minimize suffering

22 Ducks Challenge Experiments and Sample CollectionThe 120 three-day-old domestic ducklings (Jingding duck)were purchased from the Chinese Waterfowl GermplasmResource Pool (Taizhou China) RT-PCR was used to makesure that the ducklings had not been exposed to DHVprevious to our study [16]Then the ducklingswere randomlydivided into three groups the 40 ducklings were injectedwith 04 mL of DHV-1(ELD50 10minus4602ml) according toour earlier trials [17 18] and the 40 ducklings were injectedwith 04 mL of poly I C (05 mgmL Invivogen CaliforniaUSA) another 40 treated with normal saline (as uninfectedcontrols) Injection dose and injection method are consistentwith our earlier trials [18] Besides the ducklings afterthe infection showed the typical symptoms of hepatitis bycarrying the DHV-1 virus and the relative results have beenpublished [17] In this study at various times (0 4 8 1224 36 48 72 and 96 hours post-infection (hpi) threebirds in each group were euthanized by injecting sodiumpentobarbital (150mgkg) and killed by exsanguination TotalRNA was extracted from liver and kidney using the Trizol

reagent (Invitrogen California USA) The results of clinicalsymptoms and autopsy were recorded

Additionally five healthy three-day-old domestic ducks(Jingding duck Anas platyrhynchos) were purchased fromthe Chinese Waterfowl Germplasm Resource Pool (TaizhouChina) Tissues including liver spleen lung heart kidneythymus breast muscle and leg muscle were obtained aftereuthanasia that the ducklings were immediately anesthetizedwith sodium pentobarbital (intraperitoneal injection 150mgkg) and killed by exsanguination These birds werereferred to as morbid ducklingsThe tissues were snap-frozenin liquid nitrogen immediately and stored at -80∘C

23 RNA Extraction and Cloning of du SERPINA1 cDNATotal RNA was isolated from the liver of ducks with TRIzol(Invitrogen) according to the manufacturerrsquos instructionsand the quality of the isolated RNA was assessed by visu-alizing the ribosomal RNA bands after electrophoresis ona 10 agarose gel The PrimeScript 1st Strand cDNASynthesis Kit (TAKARA Dalian China) was used accordingto the manufacturerrsquos instructions with 1 120583g of total RNAas a template to produce cDNA Then the polymerase chainreaction (PCR) amplification was conducted using LA Taq(TAKARA) with the following conditions 94∘C for 5 min35 cycles of 94∘C for 30 s 50∘C for 30 s and 72∘C for 1min 30s followed by one cycle of 72∘C for 10 min Rapidamplification of cDNA ends (RACE) was used to obtain the5rsquo and 3rsquo ends of du SERPINA1 using the SMART RACEcDNAamplification protocol (ClontechMountainViewCAUSA) and the 3rsquo-Full RACE Kit (TaKaRa Dalian China)respectively Gene-specific primers used for the amplificationof RACE cDNA fragments were designed based on theobtained SERPINA1 nucleotide sequence The sequences ofdu SERPINA1was submitted to GenBank under the accessionnumber KY471047 All the primer sequences mentionedabove are shown in Table 1

24 Bioinformatics Analysis Bioinformatic analysis of duSERPINA1 was performed using a software program likeDNASTAR theNCBIwebsite (httpwwwncbinlmnihgov) andBLAST analysis (httpsblastncbinlmnihgovBlastcgi)Multiple sequence alignment was created by AlignIR V20

BioMed Research International 3

and CLC Sequence Viewer 6 was performed to constructmultiple sequence alignments of the amino acid sequences ofdu SERPINA1 proteins The neighbor-joining phylogenetictree was constructed based on the alignment result usingthe Neighbour-Joining (NJ) algorithm within the MEGA 60program

25 Quantitative Real-Time Polymerase Chain ReactionTotal RNA was extracted from different tissues using TRIzol(Invitrogen) and 1 120583g total RNAwas used with FastQuant RTKit (With gDNase) (Tiangen Beijing China) during reversetranscription The process included an initial phase at 42∘Cfor 3 min then incubation at 42∘C 15 min and incubationat 95∘C for 3 min The cDNA was stored at -80∘C Real-time qPCRwas carried out on Applied Biosystems 7500Real-Time PCR System (ThermoFisher Scientific Waltham MAUSA) with the following program 1 cycle at 95∘C for 5 minfollowed by 40 cycles of 94∘C for 30 sec 60∘C for 30 sec and72∘C for 30 sec and a final incubation at 72∘C for 10 minRelative quantitative of gene expression was calculated usingthe 2minusΔΔCt method [19] The GAPDH gene was used as aninternal standard for relative expression levels In additionat each time point the mean ΔCt value of control ducks wasused to calibrate analysis of expression patterns in infectedducks Therefore the results calibrated by the expressionsof control ducks are displayed in the plot of du SERPINA1expression of liver and kidney after DHV-1 and poly I Cchallenge All the primers are listed in Table 1

26 Enzyme-Linked Immunosorbent Assay (ELISA) AnalysisFor ELISA analysis by using double antibody sandwichmethod to determine the levels of IFN-120572 IFN-120574 IgG and IL-8 duck blood were gathered and supernatants were collectedafter centrifugation at 3000 rpm for 20 min at 4∘C andsubjected to ELISA for detection of duck IFN-120572 IFN-120574IgG and IL8 The concentrations of IFN-120572 IFN-120574 IgG andIL8 in the samples were measured with a multifunctionalmicroplate reader (Tecan Infinite M200 PRO Switzerland)and determined by comparing the optical density of thesamples to the standard curve

27 Statistical Analysis Data were analyzed using SPSS 220and differences among tissue samples were assessedwith one-way ANOVA P values less than 005 were considered assignificant All data were processed by GraphPad Prism 50

3 Results

31 du SERPINA1 cDNASequence and Bioinformatics Analysisof SERPINA1 in Ducks The full-length cDNA sequence ofdu SERPINA1 was 1457 bp in size and contained 47-bp 5rsquoUTR 135-bp 3rsquo UTR The open reading frame was 1275 bpand encoded a single open reading frame of 424 aminoacid (aa) residues without a stop codon The sequence hasbeen submitted to GenBank (GenBank accession numbersKY471047)

Comparing of the full-length aa sequence of du SER-PINA1 to the SERPINA1 gene of other species by CLCSequence Viewer 6 multiple alignments and amino acid

sequence are shown in Figure 1(a) The duck SERPINA1shared high similarity with SERPINA1 proteins from othervertebrates Compared to chickens protein from theGenBankdatabase the du SERPINA1 protein had identities of 99 Acondensed phylogenetic tree was constructed based on thederived amino acid sequences of SERPINA1 of organisms(Figure 1(b))The overall tree topology revealed the followingthree major groups bird mammal and fish

32 Tissue-Specific Expression Profile of du SERPINA1 GeneReal-time qPCR was carried out to determine the tissue-specific expression profile of du SERPINA1 gene in healthyducks The results showed that du SERPINA1 mRNA waswidely expressed in all 8 tissues tested (liver spleen lungheart kidney thymus breast muscle and leg muscle) Theexpression levels of du SERPINA1 mRNA were more signif-icant higher in lung and liver tissues whereas the expressionlevel in other tissues was extremely low (Figure 2)

33 The Expression of Cytokine following DHV-1 and PolyIC Treatment In order to investigate the changes of cellularimmunity and humoral immunity in ducklings during DHV-1 and poly IC infection the ELISA analysis was used todetect the changes of serum IgG IFN-120572 and other cytokinesThe results showed that the cytokines including IFN-120572and IFN-120574 generally indicated the fluctuating trend whichperformed the trend of first increasing then decreased andthen increased (Figures 3(a) and 3(b)) while IL8 and IgG didnot change much as a whole (Figures 3(c) and 3(d))

34 du SERPINA1 Expression after DHV-1 and Poly ICChallenge To further define the expression change of SER-PINA1 during DHV-1 or poly I C treatment time-dependentexpression levels in the liver and kidney after DHV-1 or polyI C challenge were characterized (Figure 4) In the liver uponDHV-1 treatment expression of du SERPINA1 increased at 12h post-infection (hpi) and reached peak at 48 hpi after thispeak expression of du SERPINA1 then progressively droppedIn the poly I C treatment group expression of du SERPINA1increased at 36 hpi reaching a peak at 72 hpi and thendecreased dramatically (Figure 4(a)) The expression of duSERPINA1 in kidneywas increased immediately at 4 hpi withDHV-1 and poly I C (Figure 4(b))

4 Discussion

SERPINA1 also known as Alpha-1 anti-trypsin (AAT) isa multifunctional protein with proteinase inhibitory anti-inflammatory and cytoprotective properties Some studieshave shown AAT could prevent the lethality of TNF orendotoxin in mice [20] It is also illustrated in humanthat AAT can decrease the release of TNF-120572 from LPSstimulated and unstimulated monocytes in vitro Besides inmost circulation AAT was synthesized in the liver and whensuffering an acute phase of inflammation or infection theAAT was released rapidly [21]

In healthy ducks the results of tissue-specific transcrip-tional profile of du SERPINA1 showed the uneven expressionin different tissues The SERPINA1 mRNA expression in


416421418418424425433414

295300297295297299313293

216221218215218220234214

136141138135138140154134

62676461646680

80604020

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

100 120 140 160

320

240220200180

260 280 300

380360340

420 440

54

374379376375376378392372

400

(a)

100

01

99

99

89

99

100

100

100 Homo sapiens SERPINA1Gorilla gorilla SERPINA1

Bos taurus SERPINA1Sus scrofa SERPINA1

Rattus norvegicus SERPINA1

Gallus gallus SERPINA1Anas platyrhynchos SERPINA1

Xenopus tropicalis SERPINA1Danio rerio SERPINA1

Ctenopharyngodon idella SERPINA1

Lonchura striata domestica SERPINA1

(b)

Figure 1 (a) Multiple alignments of SERPINA1 protein sequences from human mouse cow pig xenopus duck chicken and zebrafish(b) Phylogenetic analysis of avian mammalian and fish SERPINA1 was carried out The tree was constructed by the neighbor-joining treemethod using amino acid sequences aligned with MEGA6 The bar indicates the bootstrap value () The species names and GenBankaccession numbers of the SERPINA1 sequences shown are as follows Homo sapiens NM 0011277071 Gorilla gorilla gorilla XM 0190097481Bos taurus NM 1738822 Sus scrofa NM 0013489421 Rattus norvegicus NM 0225192 Lonchura striata domestic NM 0011277061Gallus gallus NM 0012774931 Anas platyrhynchos KY471047 Xenopus tropicalis NM 0010112751 Danio rerio NM 0010777581Ctenopharyngodon idella EU6214051


Heart

Liver

Splee

nLung

Kidney

Thym

us

Breast m

uscle

Leg muscl

e0

20

40

60

80

Rela

tive e

xpre

ssio

n

b

a

bc

a

bc bc c c

lowast

lowast

Figure 2 du SERPINA1 mRNA expression in various tissues (liver spleen lung heart kidney thymus breast muscle and leg muscle) Allassays were repeated at least three times and data are shown as mean plusmn SE (n = 5) from one representative experiment The expression ofSERPINA1 was normalized to GAPDH Different letters showed significant difference (p lt 005)

435

325

215

105

0h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(a)

353

252

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(b)

25

IgG

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(c)

25

IL-8

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(d)

Figure 3 Effect of DHV-1 and poly IC treatment on the cytokine in the duckings All assays were repeated at least three times and data areshown as mean plusmn SE (n = 5) from one representative experiment The expression is shown for (a) IFN-120572 (b) IFN-120574 (c) IgG and (d) IL-8

liver was significantly higher than in other tissues sup-porting the fact that SERPINA1 was attributed to differentroles in physiology particularly including immune responsesassociated with liver disease which was similar with theexpression pattern of SERPINA1 gene in human and mice[22 23] Besides the high expression of SERPINA1 was alsoexisted in lung which was consistent with previous studies

that SERPINA1 may attenuate inflammation in ventilator-induced lung injury by modulating inflammation-relatedprotein expression [24 25]

Cytokines can regulate the physiological functions ofleukocytes mediate inflammatory responses participate inimmune responses and repair tissue In order to further studythe changes of host immune and inflammatory response


0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)

Figure 4 Expression of du SERPINA1 in liver and kidney afterDHV-1 or poly IC challengeDifferent letters and asterisks showed a significantdifference (p lt 005) The expression is shown for (a) liver and (b) kidney

caused by DHV-1 and poly I C infection the levels ofcytokines in serum were tested during the process ofinfection IFN-120572 IFN-120574 as immunomodulatory pleiotropicfactors have been used as an indicator of cell-mediatedimmunity in infected organisms [26] and IL8 acts as aninflammatory cytokine and participates in the hosts inflam-matory response In this study IFN-120572 and IFN-120574 got peakedat the early stage after DHV-1 and poly IC infection and thechanges of IL8 and IgG were not significant after infectionwith DHV-1 and poly IC [27 28] These results revealed thecells of the immune system was directly damage after viralinfection and T-cell immunity played a major role in theprocess of DHV-1 and poly IC infection supplementing byB-cell immunity

Subsequently in order to explore the dynamic expressionpattern of du SERPINA1 in ducks during viral infection thedu SERPINA1 mRNA expression after DHV-1 or poly I Cchallenge was detected Previous studies have showed thatDHV-1 rather than other DHV types could specifically infectduck embryo liver and duck embryo kidney cells [29 30]and in this study liver and kidney tissues were selected to becandidate tissues During the early period of viral injectionthe expression of SERPINA1 was no significant difference inthe liver while it was rapidly increased in the kidney tissuewhichmay be related that during suffering the infection of theviral antigen the hostrsquos various non-specific defenses such asnatural killer lymphocyte cell immunity secrets more IFN-120572 and IFN-120574 [31] to regulate the expression of SERPINA1gene regulation pathway which lead to an increase in earlySERPINA1 gene expression After achieving the peak theexpression of du SERPINA1 decreased to a normal levelwhich may be prevented excessive inflammatory reactionIn pig [32] when the expression of du SERPINA1 was up-regulated after PCV2 infection the immune factor was trans-ported to the immune related tissues through the circulationof blood which prevented excessive inflammatory reactionand maintained the integrity and normal function of theimmune tissue With the increase of age the immune systemof ducklings is becoming more and more adaptive Studies

have shown that the immune level of 7-day-old chicken iscomparable to that of adult chickens [6] and the matureimmune system will produce a large number of cytokines toresist the attack of virus These results suggested SERPINA1might be associated with the host defence response againstviral infection The specific mechanism is unclear but mayautocrine regulation of SERPINA1 mRNA synthesis whenvirus invasion

Previous studies have proved that SERPINA1 has a vitalrole in protect host tissue against injuring at sites of inflam-mation Now increasing evidence showed that SERPINA1(AAT) may exhibit biological activity independent of itsprotease inhibitor function [33 34] From our results wespeculated that when being attacked by virus the hostneeds more SERPINA1 which is consistent with the previousresearch results To conclude our results provide a betterunderstanding of du SERPINA1 function in immunity duringviral infection

5 Conclusions

In this study we first cloned and characterized the gene induck and demonstrated that the du SERPINA1 gene sharedhigh similarity with SERPINA1 proteins from other verte-brates Transcriptional analyses showed ubiquitous expres-sion of SERPINA1 gene in eight examined tissues Expres-sion analyses showed that SERPINA1 gene was significantlyupregulated in vivo after DHV-1 or poly I C stimulationTaken together our results provide a better understanding thedynamic expression change of SERPINA1 gene against virusand thereby provide a theoretical basis for future immunepathological studies

Abbreviations

RT-PCR Reverse transcription polymerase chainreaction

RACE Rapid amplification of cDNA endsORF Open reading frame


Real-time qPCR Real-time quantitative polymerase chainreaction

DHV-1 Duck hepatitis virusAAT Alpha-1 anti-trypsinESTs Expressed sequence tagsSSH Suppression subtractive hybridizationNJ Neighbour-JoiningPoly IC Polyinosinic acidpolycytidylicELISA Enzyme-linked immunosorbent assay

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by grants from the ModernAgro-industry Technology Research System (CARS-43-3)the National Key Technology RampD Program of JiangsuProvince (E2014381) the National Natural Science Foun-dation of China (31601931) Jiangsu Natural Science Foun-dation (BK20150439) the Natural Science Foundation ofChina (31572385) and Jiangsu University Natural Foundation(16KJB230002)The funders had no role in the design of thestudy and collection analysis and interpretation of data andin writing the manuscript

References

[1] P G W Gettins ldquoSerpin structure mechanism and functionrdquoChemical Reviews vol 102 no 12 pp 4751ndash4803 2002

[2] I M Nita D Serapinas and S M Janciauskiene ldquo1205721-Antitrypsin regulates CD14 expression and soluble CD14 levelsin human monocytes in vitrordquo The International Journal ofBiochemistry amp Cell Biology vol 39 no 6 pp 1165ndash1176 2007

[3] D A Lomas and R W Carrell ldquoSerpinopathies and theconformational dementiasrdquoNature Reviews Genetics vol 3 no10 pp 759ndash768 2002

[4] P K Arora H C Miller and L D Aronson ldquo1205721-Antitrypsin isan effector of immunological stasisrdquo Nature vol 274 no 5671pp 589-590 1978

[5] A Churg R D Wang C Xie and J L Wright ldquo120572-1-antitrypsin ameliorates cigarette smoke-induced emphysema inthe mouserdquo American Journal of Respiratory and Critical CareMedicine vol 168 no 2 pp 199ndash207 2003

[6] I Nita C Hollander U Westin and S-M Janciauskiene ldquoPro-lastin a pharmaceutical preparation of purified human alpha1-antitrypsin blocks endotoxin-mediated cytokine releaserdquo Res-piratory Research vol 6 article 12 2005

[7] Y H Huang N Li D W Burt and F Wu ldquoGenomic researchand applications in the duck (Anas platyrhynchos)rdquo WorldrsquosPoultry Science Journal vol 64 no 3 pp 329ndash341 2008

[8] Q Xu Y Chen W M Zhao et al ldquoDNA methylation andregulation of the CD8A after duck hepatitis virus type 1infectionrdquo PLoS ONE vol 9 no 2 Article ID e88023 2014

[9] Q Xu Y Chen W M Zhao et al ldquoThe CD8120572 gene in duck(Anatidae) cloning characterization and expression duringviral infectionrdquoMolecular Biology Reports vol 42 no 2 pp 431ndash439 2015

[10] Q Xu Y Chen Y Zhang et al ldquoMolecular cloning andexpression analysis of ferritin heavy polypeptide 1 gene fromduck (Anas platyrhynchos)rdquoMolecular Biology Reports vol 41no 9 pp 6233ndash6240 2014

[11] Q Xu Y Chen Y Y Tong et al ldquoIdentification and expressionanalysis of the leukocyte cell-derived chemotaxin-2 (LECT2)gene in duck (Anas platyrhynchos)rdquo Gene vol 533 no 1 pp280ndash285 2014

[12] B Wang Y Chen C Mu et al ldquoIdentification and expressionanalysis of the interferon-induced protein with tetratricopep-tide repeats 5 (IFIT5) gene in duck (Anas platyrhynchosdomesticus)rdquo PLoS ONE vol 10 no 3 Article ID e01210652015

[13] C Song S Yu Y Duan et al ldquoEffect of age on the pathogenesisof DHV-1 in Pekin ducks and on the innate immune responsesof ducks to infectionrdquo Archives of Virology vol 159 no 5 pp905ndash914 2014

[14] M Pan X Yang L Zhou et al ldquoDuck hepatitis a virus possessesa distinct type IV internal ribosome entry site element ofpicornavirusrdquo Journal of Virology vol 86 no 2 pp 1129ndash11442012

[15] L I Xiu XUQi Y Zhang et al ldquoConstruction of a suppressionsubtractive hybridization cDNA library to screen differentiallyexpressed genes from duck liver infected duck hepatitis virusrdquoChinese Journal of Animal amp Veterinary Sciences vol 43 no 2pp 211ndash219 2012

[16] M Yang A Cheng M Wang and H Xing ldquoDevelopment andapplication of a one-step real-time Taqman RT-PCR assay fordetection of Duck hepatitis virus type1rdquo Journal of VirologicalMethods vol 153 no 1 pp 55ndash60 2008

[17] Q Xu Y Chen W M Zhao et al ldquoDNA methylation andregulation of the CD8A after duck hepatitis virus type 1infectionrdquo PLoS ONE vol 9 no 2 2014

[18] Q Xu T Gu R Liu et al ldquoFTH1 expression is affected bypromoter polymorphism andnotDNAmethylation in responseto DHV-1 challenge in duckrdquo Developmental amp ComparativeImmunology vol 79 pp 195ndash202 2018

[19] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[20] C Libert W Van Molle P Brouckaert and W Fiers ldquo1205721-antitrypsin inhibits the lethal response to TNF in micerdquo TheJournal of Immunology vol 157 no 11 pp 5126ndash5129 1996

[21] R Mahadeva and D A Lomas ldquoAlpha1-antitrypsin deficiencycirrhosis and emphysemardquo Thorax vol 53 no 6 pp 501ndash5051998

[22] M Brantly T Nukiwa and R G Crystal ldquoMolecular basis ofalpha-1-antitrypsin deficiencyrdquo American Journal of Medicinevol 84 no 6 pp 13ndash31 1988

[23] G D Kelsey S Povey A E Bygrave and R H Lovell-BadgeldquoSpecies- and tissue-specific expression of human alpha 1-antitrypsin in transgenicmicerdquoGenes ampdevelopment vol 1 no2 pp 161ndash171 1987

[24] R Joshi A Heinz Q Fan S Guo et al ldquoRole for Cela1 inpostnatal lung remodeling and alpha-1 antitrypsinndashdeficientemphysemardquo American Journal of Respiratory Cell and Molec-ular Biology vol 59 no 2 pp 167ndash178 2018


[25] H Zhu J He J Liu et al ldquoAlpha 1-antitrypsin amelioratesventilator-induced lung injury in rats by inhibiting inflam-matory responses and apoptosisrdquo Experimental Biology ampMedicine vol 243 no 1 2017

[26] T H M Ottenhoff and T Mutis ldquoRole of cytotoxic cells in theprotective immunity against and immunopathology of intracel-lular infectionsrdquo European Journal of Clinical Investigation vol25 no 6 pp 371ndash377 1995

[27] S R McColl C J Roberge B Larochelle and J Gosselin ldquoEBVinduces the production and release of IL-8 and macrophageinflammatory protein-1120572 in human neutrophilsrdquoThe Journal ofImmunology vol 159 no 12 pp 6164ndash6168 1997

[28] M Wahlgren K Berzins P Perlmann and M Persson ldquoChar-acterization of the humoral immune response in Plasmodiumfalciparum malaria II IgG subclass levels of anti-P falciparumantibodies in different serardquo Clinical amp Experimental Immunol-ogy vol 54 no 1 pp 135ndash142 1983

[29] E F Kaleta ldquoDuck viral hepatitis type 1 vaccinationmonitoringof the immune responsewith amicroneutralisation test in pekinduck embryo kidney cell culturesrdquo Avian Pathology vol 17 no2 pp 325ndash332 1988

[30] P R Woolcock ldquoAn assay for duck hepatitis virus type I in duckembryo liver cells and a comparison with other assaysrdquo AvianPathology vol 15 no 1 pp 75ndash82 1986

[31] A Schwarting G Tesch K Kinoshita R Maron H L Weinerand V R Kelley ldquoIL-12 drives IFN-120574-dependent autoimmunekidney disease in MRL-Fas(lpr) micerdquoThe Journal of Immunol-ogy vol 163 no 12 pp 6884ndash6891 1999

[32] H Liu Expression Regulation of SERPINA1 And MRC1 Genesand Their Association with Anti-PCV2 Infection in Pig Shan-dong Agricultural University 2016

[33] S Janciauskiene S Larsson P Larsson R Virtala L Janssonand T Stevens ldquoInhibition of lipopolysaccharide-mediatedhuman monocyte activation in vitro by 1205721-antitrypsinrdquo Bio-chemical and Biophysical ResearchCommunications vol 321 no3 pp 592ndash600 2004

[34] S M Janciauskiene I M Nita and T Stevens ldquoAlpha1-antitrypsin old dog new tricks 1205721- antitrypsin exerts in vitroanti-inflammatory activity in human monocytes by elevatingcAMPrdquoThe Journal of Biological Chemistry vol 282 no 12 pp8573ndash8582 2007

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Table 1 Primers used in the study

Primer name Primer sequence (5rsquo997888rarr3rsquo) Annealing temperature (∘C) Applicationdu SERPINA1-F ATGAAAATGAAGTCTGCACTG 55 RT-PCRdu SERPINA1-R CTAGCCTACCTTGAGAGTTGGGdu SERPINA1 5rsquo Router GTTCTTGTTACCTTCCTCCT 60 5rsquo RACE-PCRdu SERPINA1 5rsquo Inner GGAGAGGCTCAAAGGAdu SERPINA1 3rsquo Router AGGTGACCGCCTTATGGTTTCC 60 3rsquo RACE-PCRdu SERPINA1 3rsquo Inner AATAATGCTGACCTCTCTGGAGTGGCqdu SERPINA1-F AATACTAGCCAGCAAAACGAA 60 RT-qPCRqdu SERPINA1-R TGAGCCAAGTTTTCACCTTCGAPDH-F TGCTAAGCGTGTCATCATCT 60 RT-qPCRGAPDH-R AGTGGTCATAAGACCCTCCA

of SERPINA1 using a suppression subtractive hybridization(SSH) cDNA library of 3-day-old ducklings treated withDHV-1 [15] Our study showed that during DHV-1 and polyIC infection the expression of SERPINA1 mRNA was up-regulated In this study we aim to expand those preliminaryresults by assessing tissue-specific gene expression and thedynamic expression change of the SERPINA1 gene againstthe virus and thereby provide a theoretical basis for futureimmune pathological studies

2 Materials and Methods

21 Ethics Approval and Consent to Participate The animalexperiment was reviewed and approved by the InstitutionalAnimal Care and Use Committee of Yangzhou University(approval number 151-2014) Procedures were performedin accordance with the Regulations for the Administrationof Affairs Concerning Experimental Animals (YangzhouUniversity China 2012) and the Standards for the Adminis-tration of Experimental Practices (Jiangsu China 2008) Wealso confirm that all efforts were made to minimize suffering

22 Ducks Challenge Experiments and Sample CollectionThe 120 three-day-old domestic ducklings (Jingding duck)were purchased from the Chinese Waterfowl GermplasmResource Pool (Taizhou China) RT-PCR was used to makesure that the ducklings had not been exposed to DHVprevious to our study [16]Then the ducklingswere randomlydivided into three groups the 40 ducklings were injectedwith 04 mL of DHV-1(ELD50 10minus4602ml) according toour earlier trials [17 18] and the 40 ducklings were injectedwith 04 mL of poly I C (05 mgmL Invivogen CaliforniaUSA) another 40 treated with normal saline (as uninfectedcontrols) Injection dose and injection method are consistentwith our earlier trials [18] Besides the ducklings afterthe infection showed the typical symptoms of hepatitis bycarrying the DHV-1 virus and the relative results have beenpublished [17] In this study at various times (0 4 8 1224 36 48 72 and 96 hours post-infection (hpi) threebirds in each group were euthanized by injecting sodiumpentobarbital (150mgkg) and killed by exsanguination TotalRNA was extracted from liver and kidney using the Trizol

reagent (Invitrogen California USA) The results of clinicalsymptoms and autopsy were recorded

Additionally five healthy three-day-old domestic ducks(Jingding duck Anas platyrhynchos) were purchased fromthe Chinese Waterfowl Germplasm Resource Pool (TaizhouChina) Tissues including liver spleen lung heart kidneythymus breast muscle and leg muscle were obtained aftereuthanasia that the ducklings were immediately anesthetizedwith sodium pentobarbital (intraperitoneal injection 150mgkg) and killed by exsanguination These birds werereferred to as morbid ducklingsThe tissues were snap-frozenin liquid nitrogen immediately and stored at -80∘C

23 RNA Extraction and Cloning of du SERPINA1 cDNATotal RNA was isolated from the liver of ducks with TRIzol(Invitrogen) according to the manufacturerrsquos instructionsand the quality of the isolated RNA was assessed by visu-alizing the ribosomal RNA bands after electrophoresis ona 10 agarose gel The PrimeScript 1st Strand cDNASynthesis Kit (TAKARA Dalian China) was used accordingto the manufacturerrsquos instructions with 1 120583g of total RNAas a template to produce cDNA Then the polymerase chainreaction (PCR) amplification was conducted using LA Taq(TAKARA) with the following conditions 94∘C for 5 min35 cycles of 94∘C for 30 s 50∘C for 30 s and 72∘C for 1min 30s followed by one cycle of 72∘C for 10 min Rapidamplification of cDNA ends (RACE) was used to obtain the5rsquo and 3rsquo ends of du SERPINA1 using the SMART RACEcDNAamplification protocol (ClontechMountainViewCAUSA) and the 3rsquo-Full RACE Kit (TaKaRa Dalian China)respectively Gene-specific primers used for the amplificationof RACE cDNA fragments were designed based on theobtained SERPINA1 nucleotide sequence The sequences ofdu SERPINA1was submitted to GenBank under the accessionnumber KY471047 All the primer sequences mentionedabove are shown in Table 1

24 Bioinformatics Analysis Bioinformatic analysis of duSERPINA1 was performed using a software program likeDNASTAR theNCBIwebsite (httpwwwncbinlmnihgov) andBLAST analysis (httpsblastncbinlmnihgovBlastcgi)Multiple sequence alignment was created by AlignIR V20






3 Results







4 Discussion




416421418418424425433414

295300297295297299313293

216221218215218220234214

136141138135138140154134

62676461646680

80604020

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

100 120 140 160

320

240220200180

260 280 300

380360340

420 440

54

374379376375376378392372

400

(a)

100

01

99

99

89

99

100

100








(b)



Heart

Liver

Splee

nLung

Kidney

Thym

us

Breast m

uscle

Leg muscl

e0

20

40

60

80

Rela

tive e

xpre

ssio

n

b

a

bc

a

bc bc c c

lowast

lowast


435

325

215

105

0h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(a)

353

252

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(b)

25

IgG

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(c)

25

IL-8

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(d)






0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)






5 Conclusions


Abbreviations






Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018







3 Results







4 Discussion




416421418418424425433414

295300297295297299313293

216221218215218220234214

136141138135138140154134

62676461646680

80604020

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

100 120 140 160

320

240220200180

260 280 300

380360340

420 440

54

374379376375376378392372

400

(a)

100

01

99

99

89

99

100

100








(b)



Heart

Liver

Splee

nLung

Kidney

Thym

us

Breast m

uscle

Leg muscl

e0

20

40

60

80

Rela

tive e

xpre

ssio

n

b

a

bc

a

bc bc c c

lowast

lowast


435

325

215

105

0h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(a)

353

252

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(b)

25

IgG

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(c)

25

IL-8

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(d)






0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)






5 Conclusions


Abbreviations






Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



416421418418424425433414

295300297295297299313293

216221218215218220234214

136141138135138140154134

62676461646680

80604020

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

HumanMouse

CowPig

XenopusDuck

ChickenZebrafish

100 120 140 160

320

240220200180

260 280 300

380360340

420 440

54

374379376375376378392372

400

(a)

100

01

99

99

89

99

100

100








(b)



Heart

Liver

Splee

nLung

Kidney

Thym

us

Breast m

uscle

Leg muscl

e0

20

40

60

80

Rela

tive e

xpre

ssio

n

b

a

bc

a

bc bc c c

lowast

lowast


435

325

215

105

0h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(a)

353

252

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(b)

25

IgG

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(c)

25

IL-8

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(d)






0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)






5 Conclusions


Abbreviations






Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Heart

Liver

Splee

nLung

Kidney

Thym

us

Breast m

uscle

Leg muscl

e0

20

40

60

80

Rela

tive e

xpre

ssio

n

b

a

bc

a

bc bc c c

lowast

lowast


435

325

215

105

0h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(a)

353

252

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

IFN

-(p

gm

l)

(b)

25

IgG

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(c)

25

IL-8

(pg

ml) 2

151

050

h

DHVPloy IC

4 8 12 24 36 48 72 960

(d)






0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)






5 Conclusions


Abbreviations






Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0 4 8 12 24 36 48 72 96

Liver

Post infection time

DHV-1poly IC

0

5

10

15

20

Relat

ive e

xpre

ssio

n

minus5

(a)

0 4 8 12 24 36 48 72 96

90190290390

100015002000

2500300035004000 Kidney

Post infection time

Rela

tive e

xpre

ssio

n

DHV-1poly IC

minus10

lowast

lowast

(b)






5 Conclusions


Abbreviations






Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





Data Availability




Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Dynamic Expression Pattern of SERPINA1 Gene from Duck ...downloads.hindawi.com/journals/bmri/2019/1321287.pdf · Dynamic Expression Pattern of SERPINA1 Gene from Duck (Anas platyrhynchos)