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目的构建人Ro60cDNA的杆状病毒表达载体。方法采用逆转录-PCR技术从Hela细胞RNA中扩增Ro60cDNA,定向插入杆状病毒转移载体pFastBacHTc,转化大肠杆菌DH10Bac进行转座,提取重组Bacmid,通过蓝白斑筛选和PCR进行鉴定。结果成功从Hela细胞RNA中扩增出1.56kbRo60,所克隆的Ro60cDNA与已公布的序列完全符合,证明本克隆为Ro60cDNA的精确拷贝,并证实其目的基因与重组载体发生了特异转座和病毒重组,成功地构建了重组杆状病毒表达载体Bacmid-Ro60。结论本实验成功克隆了Ro60cDNA并构建了其杆状病毒表达载体,为进一步表达Ro60蛋白和研究此抗原在红斑狼疮的发病机制奠定了基础。
Objective To construct the baculovirus expression vector of human Ro60 cDNA. Methods Ro60 cDNA was amplified from RNA of Hela cells by RT-PCR and inserted into baculovirus transfer vector pFastBacHTc. The recombinant plasmid was transformed into E. coli DH10Bac for transposition. The recombinant Bacmid was isolated and identified by blue-white screening and PCR. Results A total of 1.56 kbRo60 was successfully amplified from Hela cell RNA. The Ro60 cDNA cloned was completely consistent with the published sequence, which proved that this clone was an exact copy of Ro60 cDNA and confirmed that the target gene was specifically transposed with the recombinant vector and virus recombination , Successfully constructed a recombinant baculovirus expression vector Bacmid-Ro60. Conclusion The experiment successfully cloned Ro60 cDNA and constructed its baculovirus expression vector, which laid the foundation for the further expression of Ro60 protein and the study of the pathogenesis of this antigen in lupus.