摘要
用限制性内切酶从克隆质粒pUC—s中回收猪传染性胃肠炎病毒中国分离株,TH-98株完整的病毒保护性抗原基因,即S基因。按照预定的阅读框架,将S基因克隆于供体质粒pFASTBACHTb的EcoRI、PstI多克隆位点上,并进行酶切鉴定,得到阳性重组质粒BAC—S。用DH10BAC对BAC—S进行转位,获得重组质粒pBAC—S。根据S基因酶切位点分析结果及杆状病毒(Bac-ulovirus)转位区结构特点,用相应的限制性内切酶进行酶切鉴定,并用特异性和通用引物进行PCR分析,证明该重组质粒为含有TH-98株完整S基因的可直接在昆虫细胞进行表达的感染性重组质粒。
The complete S gene from Chinese isolate TH-98 of transmissible gastroenteritisvirus (TGEV) was digested from the cloned pUC-S plasmid with suitable restriction enzyme (RE). The 4. 4 kb S gene carrying initiation and termination codons was subcloned EcoRI, PstI multiclonal sites of pFASTBACHTb donor plasmid according to the appropriate open reading frame(ORF). The recombinant BAC-S was identified with RE. Then,transpositionof BAC-S was accomplished with DH10BAC competent cell,and the product of it was analyzedboth by nested PCR with specific as well as general primers and corresponding RE on the basis of physical map of S gene and characteristics of transposition region of Bacmid DNA. The results demonstrated that the purified recombinant transfectious plasmid, pBACSencoding the S gene from TH-98 isolate of TGEV could be expressed directly in insect cells.
出处
《东北农业大学学报》
CAS
CSCD
2002年第4期359-363,共5页
Journal of Northeast Agricultural University
基金
黑龙江省科委"九五"攻关项目部分
