H5亚型不同分支禽流感病毒HA基因混编文库的构建  

Establishment of Shuffling PCR method for HA gene of H5subtype avian influenza virus from different clades

作　　者：焦晨晨 王波 赵玉博 丁蕾蕾[1] 刘静 陈普成[1] 姜永萍[1] 陈化兰[1] 柳金雄[1] JIAO Chen-chen;WANG Bo;ZHAO Yu-bo;DING Lei-lei;LIU Jing;CHEN Pu-cheng;JIANG Yong-ping;CHEN Hua-lan;LIU Jin-xiong(Animal Influenza Key Laboratory of the Ministry of Agriculture,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第12期1245-1251,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：自然科学基金面上项目(32072878)。

摘　　要：为探究DNA Shuffling能否用于H5亚型不同分支禽流感病毒(AIV)HA基因混编文库的建立,以及利用该基因文库构建重组牛痘病毒文库的可行性,本研究构建了包含H5亚型AIV 2.3.4.4分支代表株A/Chicken/Guizhou/S4184/2017(H5N6)(简称GZ/S4184)和2.3.2.1d分支代表株A/Chicken/Liaoning/SD007/2017(H5N1)(简称LN/SD007)HA基因的重组质粒pRB-GZ/S4184HA和pRB-LN/SD007HA,并经PCR鉴定正确后分别作为模板经PCR扩增两种病毒的HA基因,并利用DNaseⅠ消化后,选择两种病毒10 bp~50 bp的HA基因等质量混合后作为模板,经Shuffling PCR混编后,选取弥散条带大量集中在2000 bp左右的Shuffling PCR产物为模板经特异性PCR扩增两种AIV的混编HA(mHA)基因。结果显示,获得2000 bp左右的mHA基因,且与从上述两种重组质粒扩增的HA基因片段大小基本一致。将mHA基因克隆至pRB21质粒中,构建pRB21-mHA质粒文库,并经PCR鉴定和测序分析其多样性。结果显示,随机挑选200个菌落检测pRB21-mHA质粒文库的阳性率为92.5%,且均呈现2种亲本病毒HA基因的碱基特性且均不相同,表明构建了HA基因序列呈现多样性的pRB21-mHA质粒文库。利用pRB21-mHA质粒文库转染预先感染缺陷牛痘病毒(rdVV-ΔVP37)的CV-1细胞,构建重组牛痘病毒(rVV-mHA)库,并分别经PCR、测序和间接免疫荧光试验(IFA)鉴定。结果显示,随机挑选的200个病毒噬斑,检测重组牛痘病毒库rVV-mHA的阳性率为96%;且其mHA基因编码的氨基酸序列均呈现出2种亲本病毒HA基因的氨基酸序列特性且均不相同;IFA鉴定结果显示,mHA基因均能够通过重组牛痘病毒在细胞中正确表达。以上结果表明,利用Shuffling PCR技术获得了同时呈现H5亚型AIV GZ/S4184和LN/SD007株HA基因特性的pRB21-mHA质粒文库和rVV-mHA库。本研究首次基于H5亚型不同分支AIV的HA基因,利用DNA Shuffling技术建立了Shuffling PCR方法用于构建AIV的mHA基因文库,并利用该基因文库构建重组牛痘病毒文�To explore whether DNA Shuffling can be used for the establishment of a hybrid library of different branches of H5 subtype avian influenza virus(AIV)HA genes,and the feasibility of using this gene library to construct a recombinant vaccinia virus library.In this study,HA genes of the representative strains A/Chicken/Guizhou/S4184/2017(H5 N6,GZ/S4184,Clade2.3.2.1 d)and A/Chicken/Liaoning/SD007/2017(H5 N1,LN/SD007,Clade 2.3.4.4)were constructed into pRB plasmid,designated as pRB-GZ/S4184 HA and pRB-LN/SD007 HA,respectively.The HA genes of the two viruses were amplified by using the plasmids as templates,and digested with DNase I.DNA fragments with length of 10 bp-50 bp form both virus origins were collected and mixed as templates for Shuffling PCR.Products with diffuse bands around 2000 bp were collected and mixed as templates,and the mixed HA(mHA)gene of the two AIVs was amplified by specific PCR.The results showed that mHA genes with length of around2000 bp were obtained,and the size of the HA gene fragment amplified from the above two recombinant plasmids was identical.The mHA gene was cloned into the pRB21 plasmid,and the pRB21-mHA plasmid library was constructed,which was identified by PCR and sequencing analysis.The results showed that the positive rate of 200 randomly selected pRB21-mHA plasmids was92.5%,and the mHA gene inserts in all of the positive plasmids differed from either of the two parental HA genes,indicating that the pRB21-mHA plasmid library with diverse gene sequences was successfully constructed.The pRB21-mHA plasmid library was then used to transfect CV-1 cells infected with defective vaccinia virus(rdVV-ΔVP37)to construct a recombinant vaccinia virus(rVV-mHA)library,which was identified by PCR,sequencing analysis and indirect immunofluo-rescence(IFA).The results showed that among the 200 randomly selected virus plaques,the positive rate of the recombinant vaccinia virus library(rVV-mHA)library was 96%,and the amino acid sequence of mHA differed from either of the parental HAs;IFA identifica

关 键 词：禽流感病毒 不同分支病毒 血凝素 Shuffling PCR 

分 类 号：S852.65[农业科学—基础兽医学]