猪附红细胞体MSG1部分基因片段的全基因合成及克隆表达

Cloning and expression of synthesis fragment of Mycoplasma suis MSG1 gene

通过人工合成猪附红细胞体MSG1基因的编码序列,以提高目的基因在大肠杆菌中的高效表达。根据Gen-Bank注册的AM407404的544～942氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了12条寡核苷酸片段。用重叠区扩增法(PAS)全基因合成MSG1部分目的基因,克隆入pjet1.2/blunt载体。经测序证实正确后,构建原核表达载体pET-28c/MSG1,并转化入大肠杆菌BL21(DE3)中,获得含重组表达质粒的转化子,IPTG诱导表达,并进行SDS-PAGE分析。测序、酶切鉴定等结果表明,表达载体构建成功,诱导表达后大约在16000的位置出现明显的蛋白条带。

To synthesize the fragment of Mycoplasma suis MSG1 gene,and improve the expression of the aim protein in Escherichia coli.Base on the gene sequence of the AM407404 （544-942） which registered in the GenBank,six pairs of oligonucleotides were synthesized by using E.coli biased codons.By means of PAS to synthesize the gene which encoding the MSG1 protein,cloned into the clone vector pjet1.2/blunt and confirmed by DNA sequencing.The recombinant plasmid pET-28c/MSG1 was constructed by inserting the purified MSG1 DNA fragment into expression vector pET-28c and the expressed by transforming the recombinant plasmid pET-28c/MSG1 into E.coli BL21 and induced the recombinant with IPTG.The expressing situation of fusion protein was analyzed by SDS-PAGE.The result indicated that the recombinant plasmid was constructed successfully,and a protein with about 16 000 was induced by IPTG in SDS-PAGE.