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目的 构建人N端截断型脂多糖结合蛋白 (tLBP)基因的杆状病毒表达载体 ,为后续的蛋白表达及其抗内毒素保护作用研究奠定基础。方法 采用PCR技术从人全长LBP基因cDNA中扩增长度为 6 95bp的基因片段 ,经pGEM T载体亚克隆筛选和序列分析后 ,应用Bac To Bac杆状病毒表达系统 ,基因重组和外源基因转座分别构建转移质粒pFASTBACtLBP和穿梭质粒pBacmidtLBP。结果 经PCR、琼脂糖凝胶电泳、酶切分析及序列测定 ,所克隆的基因片段为 70 0bp左右的tLBP ,并证实其目的基因重组载体发生了特异转座和病毒重组。结论 本实验成功克隆了tLBP目的基因片段并构建其杆状病毒表达载体。
Objective To construct the baculovirus expression vector of human N-terminal truncated lipopolysaccharide binding protein (tLBP) gene, which lays the foundation for further study of protein expression and its anti-endotoxin protective effect. Methods A total length of 695bp was amplified from human full-length LBP cDNA by PCR. After subcloning and sequencing of the pGEM T vector, the Bac to Bac baculovirus expression system, gene recombination and exogenous gene Transposons were constructed transfer plasmid pFASTBACtLBP and shuttle plasmid pBacmidtLBP. Results The cloned gene fragment was about 70 bp in length by PCR, agarose gel electrophoresis, enzyme digestion analysis and sequencing. The recombinant plasmid was confirmed to be specific transposition and virus recombination. Conclusion The tLBP gene fragment was successfully cloned and its baculovirus expression vector was constructed.