家蝇幼虫防御素基因Mdd Ⅰ在毕赤酵母中的表达及其抗菌活性鉴定

Expression of Musca domestica Larvae Defensin Gene Mdd Ⅰ in P.pastoris and Identification of Its Antibacterial Activity

根据家蝇幼虫防御素基因(MddⅠ)序列,设计合成含有KpnⅠ和XbaⅠ酶切位点的特异性引物,PCR扩增出MddⅠ基因全长序列。克隆并测定序列后,构建真核表达质粒pGAPZαAMddⅠ,将其在毕赤酵母(P.pastoris)GS115中分泌表达,并对表达产物进行抑菌活性分析。结果表明,Mdd 1基因在P.pastoris如中成功表达,分子量约为10.3 kDa,抑菌结果显示MddⅠ基因的真核表达产物对猪源链球菌有抑制作用。该试验成功构建了表达MddⅠ基因的P.pastoris菌株,为进一步研究MddⅠ真核表达产物的生物学活性和免疫学活性奠定了基础。

A pair of primers with restriction sites gene sequence of Musca domestica larvae defen sin n I and Xba I were designed and synthesized according to gene Mdd I, then facilitated to amplify the full -len sequence of Mdd I by polymerase chain reaction （PCR）. After cloning and sequencing, the eukaryotic express the gth ion plasmid pGAPZαA -Mdd I was constructed and then expressed in P. pastoris GS115, subsequently the antimicrobial activity of recombinant protein was analyzed. The resuh showed that the expressed protein of Mdd I was about 10. 3 kDa, which possessed antibacterial activity against swine Streptococcus. Conclusively, the recombinant P. pastoris with gene Mdd I was successfully constructed, which was a basis for further researchers in biological and immunologic activities of eukaryotic expression product of Mdd I