麻疹H基因克隆及表达条件优化

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。

Clone of hemagglutinin gene of measles and optimization of expression condition

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.