IBV单链抗体的筛选及其互补决定区序列变异分析

Screening and analysis of CDR of single chain variable fragment against infectious bronchitis virus

【目的】筛选能表达鸡传染性支气管炎病毒(IBV)单链抗体(scFv)的原核阳性克隆,并分析scFv抗原结合位点的氨基酸变化。【方法】采用RT-PCR,从IBV疫苗免疫的鸡脾脏RNA中扩增出抗体编码基因的重链可变区(VH)和轻链可变区(VL)基因。利用重组链延伸反应(SOE-PCR),将linker与VH和VL基因相连构建鸡scFv基因,并将其克隆到原核表达载体pOPE101-XP中,构建重组质粒并转入大肠杆菌表达,间接ELISA筛选原核表达的抗IBVscFv的阳性克隆,对其测序后进行Clustal.W多序列比较,分析scFv骨架区(FR)和互补决定区(CDR)氨基酸序列差异。【结果】筛选到表达IBVscFv的阳性克隆ZL.10和ZL.80;将它们的氨基酸序列与鸡胚系Gemline进行多序列比较,结果显示,ZL.10、ZL.80在CDR中易变异氨基酸占各区氨基酸的比率均在40%以上,其中VH的CDR3达90%,VL的CDR3达60%。此外,ZL.10、ZL.80在VH的CDR3区分别增加5个和7个氨基酸。FR氨基酸变异率较少,且大多均在12%以下,而FR4未发生突变。【结论】通过体外筛选原核表达产物的方法,获得了表达IBVscFv的阳性克隆ZL.10和ZL.80,其VH、VL氨基酸序列中的变异主要发生在各自的CDR,二者VH的CDR3氨基酸序列差异很大,提示这2个scFv阳性克隆针对的是IBV不同的抗原位点。

【Objective】 The study was to select scFv aginst IBV by screening in vitro to analyse changed amino acid in complementary determinant region.【Method】 Variable fragment of heavy and light chain was amplified from immunised spleen and was constructed to scFv using splice-overlap extension polymerase chain reaction（SOE-PCR） through a flexible（Gly4Ser）3 linker.The scFv gene was cloned by the prokaryotic expression vector pOPE101-XP and then transformed to E.coli JM109 to be induced expressing.Clone expressing scFv against IBV was screened by indirect ELISA.Changed amino acid sequence of every positive clone was compared with each other.【Result】 Two positive clone expressings scFv against IBV were selected by ELISA screening.The result of analysis to amino acid sequence of ZL.10 and ZL.80,compared to Germline,shows that changed amino acid sequence in CDR is more than 40%,including 90% change in CDR3 of VH and 60% change in CDR3 of VL.There are 5 increaing amino acids in CDR3 of VH of ZL.10 and 7 in same domain of ZL.80.The amino acid in FR shows less change and no change in both FR4.【Conclusion】 ZL.10 and ZL.80,expressing scFv against IBV,are obtained by ELISA screening in vitro.Changing of amino acid sequence of ZL.10 and ZL.80 mainly ocurrs in CDR,and CDR3 in VH has the biggest change.The result reveals that the antigenic sites of IBV targeted by scFv expressed in ZL.10 and ZL.80 are different.