麻疹H基因克隆及表达条件优化

Clone of hemagglutinin gene of measles and optimization of expression condition

目的克隆麻疹H蛋白基因,构建重组表达质粒,诱导表达蛋白。方法麻疹Edmonston株减毒活疫苗中提取基因组RNA,RT-PCR扩增H基因。用限制性内切酶EcoRI和HindⅢ双酶切H蛋白基因片段和pET30a(+)载体,连接后获得重组质粒MV-H-pET30a(+),转化至BL21(DE3),IPTG诱导表达。结果 RT-PCR扩增片段长度约为1900bp,与MV-H基因相符。MV-H-pET30a(+)测序结果显示:MV-H基因对码正确,核苷酸序列符合率达99.7%。SDS-PAGE结果表明,菌体中含有特异性蛋白,大小约为86kD。结论在pET30a(+)成功克隆麻疹H基因,并有目的蛋白表达。

Objective To clone hemagglutinin gene of measles （MV）, construct recombinant expression plasmid and induce expression proteins. Methods Measles virus genomic RNA was extracted from the live attenuated vaccine （Edmonston strain ） and H gene was amplified by RT-PCR. After digested by EcoR Ⅰ and Hind Ⅲ, the H ,gene was inserted into expression plasmid pET30a （ ＋ ） by means of T4 DNA ligase. Then the recombinant plasmid was transformed into E. coli BL21 （ DE3 ） , which was induced by IPTG. The expression of H protein was detected by SDS-PAGE. Results The length of amplification fragment by RT-PCR was about 1900 bp, which was correspondence with H gene of measles. The results of sequencing for MV-H-pET30a （ ＋ ） showed that the H gene had been inserted into the vector in the correct position, and the coincidence rate of nucleotide sequence reached 99.7%. The results of SDS-PAGE detection indicated that E. coli BL21 （ DE3 ） contained specific protein, with the size being about 86kD. Conclusion The sequences of H gene of measles are successfully cloned in pET30a（ ＋ ） by means of the optimized condition ,which can express interest protein.