蛇毒金属蛋白酶抑制剂基因的合成及杆状病毒表达载体的构建

Gene Synthesis and Construction of Baculovirus Expression Vector of a Snake Venom Metalloproteinase Inhibitor

研究蛇毒金属蛋白酶抑制剂(BJ46a)基因的合成及杆状病毒表达载体的构建。根据GenBank中查找的BJ46a基因序列(AF294836),将其分为20个片段,通过缓慢退火PCR法使之拼接为完整的BJ46a基因,经SacⅠ及XhoⅠ双酶切后定向克隆到载体pET-42a(+)中,PCR扩增、酶切及测序鉴定;根据测序结果用定点突变法逐一改正错误位点。随后将该基因插入到转座载体pFastBac HT C的MCS中,转化大肠杆菌DH5-αT1,以LB/AP平板筛选阳性重组子,PCR扩增、酶切鉴定,提取pFastBac HT C-BJ46a重组体进一步转化DH10Bac感受态细胞,48h后通过蓝白筛选,挑取单个白色菌落划线,证实所得菌落均为白斑,挑克隆PCR鉴定。结果表明成功构建杆状病毒重组转座载体、重组穿梭载体。含BJ46a基因的重组杆状病毒感染Sf 9昆虫细胞,获得目的蛋白的表达。BJ46a基因的合成及杆状病毒表达载体的成功构建,为进一步研究其生物学功能奠定了坚实的基础。

To investigate gene synthesis and construction of baculovirus expression vector of a snake venom metallopmteinase i nhibitor（BJ46a）, twenty fragments of BJ46a gene from GenBank（AF294836） were synthesized. Slowly annealing PCR methods were conducted to obtain the full length of BJ46a gene. This gene was cloned into vector pET-42a（ ＋ ） at Sac Ⅰ/Xho Ⅰ site. The construct was confirmed by PCR, enzyme digestion and sequence analysis. The gene constructed by this method was found to contain 6 sequence errors, which were subsequently corrected by site-directed mutagenesis. The corrected snake BJ46a gene was cloned into the multiple cloning site of pFastBac HT C vector, and then the ligation reaction was transformed into DH5α-T1 competent E. coli, followed by selecting ampicillin-resistant transformants using LB agar plate. Positive transformants were verified by both PCR and restriction analysis. The pFastBac construct was isolated and transformed into DH10Bac competent cells. After 48 hours, blue/white selection was applied. White colony was picked and restreaked, result showed pure white colonies. Recombinant DNA was analyzed by PCR. Thus, a recombinant pFastBac HT C vector and a recombinant shuttle vector （bacmid）of baculovirus were generated. The baculovial stock contained BJ46a gene were then used to infect Sf 9 cells to produce recombinant protein. The successful synthesis of snake BJ46a gene and construction of baculovirus expression vector, will be useful for protein expression and further study of its biology functions.