J亚群禽白血病Hrb-1分离株env基因克隆及gp85杆状病毒表达载体的构建

The clone and construction of recombinant baculovirus vector of avian leukosis virus subgroup J Hrb-1 strain env gene

自哈尔滨某送检患病鸡群中分离出一株病毒,经RT＿PCR检测、SPF鸡胚成纤维细胞增殖后,获取其前病毒DNA,采用依据原型毒株HPRS＿103cDNA序列设计并合成的一对引物,PCR扩增病毒的囊膜基因,连接pMD18＿T载体并转化大肠杆菌JM109,培养后提取质粒分别用HindIII,BamHI进行单酶切和双酶切鉴定,得到了阳性重组质粒pMD18＿T＿Hrb＿1/env,对其进行PstI酶切,回收包含J亚群禽白血病病毒株Hrb＿1gp85基因的997bp片段,应用BactoBac杆状病毒表达系统,将外源片段与线性化的杆状病毒载体pFastBacHTA进行连接,获得重组载体pFASTBacHTA/gp85,将该重组载体转化DH10Bac感受态细菌,在体内进行重组,经抗性和蓝白斑筛选,获得了杆状病毒重组载体Bacmid/gp85,为表达gp85并建立适于国内应用的ELISA诊断方法奠定了基础。

In this research,one strain of ALV-J named Hrb-1 was identified by reverse transcription-polymerase chain reaction(RT-PCR).This virus was propagated on the SPF CEF and harvested after 7 days' culturing. According to the sequence of prototype ALV-J virus HPRS-103 cDNA gene published,a pair of primers were designed to amplifiy part of the env gene which including gp85,gp37 and E-element by polymerade chain reaction(PCR).The env gene obtained was inserted into pMD18-T vector and identified by single and double digestion using HindIII and BamHI,one DNA fragment containing gp85 gene of avian leukosis virus subgroup J Hrb-1 strain was obtained by digestion of pMD18-T-Hrb-1/env using Pst 1,The transpositional recombinant vector pEASTBAC/gp85 was construscted by ligation with vector pFAST bACHTA/gp85,then it was transformed in E.coli DH10Bac and cultured in LB plate which contained Tet,Kan,X-gal and IPTG.The recombinant Bacmid named as Bacmid/gp85 were obtained by selection of white positive bacterial colonies.