Mhp强弱毒株P97基因的克隆与序列测定

Molecular Cloning and Comparison between P97 Gene of Virulent and Attenuated Strains of Mycoplasma Hyopneumoniae

利用聚合酶链式反应(PCR)技术扩增出猪肺炎支原体弱毒株R659及强毒株F19的P97基因,PCR产物经纯化后,克隆至载体pGEM-Teasyvector,转化DH5α感受态细胞,筛选阳性克隆,提取质粒,对目的片段测序,结果表明:R659的P97全基因大小为3416bp,F19的P97全基因大小为3329bp,强弱毒株间核苷酸同源性为96.9%,弱毒株R659与标准株232A核苷酸同源性为95.6%,强毒株F19与232A株核苷酸同源性为95.1%。

The attempt of this research was to probe the secret of attenuation course in vivo of R659 by studying the difference existing in gene clone and sequence of virulent and attenuated strains of Mhp. In this study, mycoplasma hyopneumoniae was cultured in pm media, and the Mhp DNA was extracted. Then P97 genes of R659 and F19 were amplified by PCR. The target fragment was purified, then was cloned into pGEM-T easy vector by T-A ligation. Then transformed the ligation products into the DH5α competent cells. Consequently, the recombinant plasmid was amplified and extracted from the transformants while the inserting of the P97 fragment was tested by PCR. Sequencing the P97 fragment showed that the whole length of P97 gene (about 3.4 kb) were cloned successfully. Compared with the published P97 sequences (232A strain), the homology of R659 and F19 in nucleotides are 95.6%, 95.1% respectively. Between R659 and F19, the homology is 96.9%. Such result suggested that the obvious morbidity disparity lies in virulent and attenuated strains in R1 repeat sequence of P97 gene. The successful cloning P97 gene paved a way for developing the gene engineering vaccine of Mhp.