Metarhizium anisopliae var. Anisopliae Pr1A基因的克隆表达及抗血清研究

采用RT-PCR方法从本实验室分离筛选到的金龟子绿僵小孢变种Metarhizium anisopliae vat．anisopliae中,扩增得到PrlA基因全长,此基因全长为1242bp,经Blastn分析此基因序列与M．anopliae的PrlA基因(M73795)同源率为98%。以pET- 22b( )为基础载体,构建pET-PrlA重组表达载体,在大肠杆菌(Escherichia．coli)BL 21(DE3)中进行表达。经SDS—PAGE分析,获得了约42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63．2%。将表达的PrlA蛋白切胶回收后制备成抗原,免疫家兔4次后,采血收集抗血清,用ELISA测定效价为1/10000。结果表明,获得的抗体可用于更进一步的研究,将有利于我们进一步了解M．anisopliaeis的侵染机理,弄清楚各Pr蛋白酶的作用方式和对寄主的选择优势,提高生防控制的有效性。

Cloning and Expressing Pr1A Gene of the Entomopathogene fungus Metarhizium anisopliae var. anisopliae and Researching Its Antiserum

The Pr1A gene was amplified by RT-PCR from Metarhizium anisopliae var.anisopliae,The whole length of this gene was 1242bp, and the nucleotide sequence of the gene was 98% similarity to that of the M.anisopliae accessed in GenBank(M73795).The gene was subcloned into prokaryon expression vector pET-22b( ),transformed this recombinant expression plasmid into E.coli strain BL 21(DE3) and effective expressed.The SDS-PAGE analysis indicated that the recombinant protein was about 42kDa which is same to the reported article.The expression level of recombinant protein was about 63.2% of the whole expressed proteins.After SDS-PAGE,recovered the protein of Pr1A and made anti- gen,injected rabbits by antigen.After fourth injection,blood was harvested by carotid artery and the antiserum was made from it.ELISA proved that the titres of the antiserum was 1/10000.The result indicated that the antibody of Pr1A protein could be used in further researches.It could help us to further understand the infect mechanism of Metarhizium anisopliae,make sure of every Pr proteins' mode of action and the predominance of selecting host,so could improve the efficiency of biological controlling.