【作者】 刘茂军；

【导师】 聂向庭； 邵国青； 张映；

【作者基本信息】 山西农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 猪肺炎支原体是引起猪气喘病的主要病原。黏附因子是支原体致病的关键因子。本研究根据已报道的序列为参考，设计一对带有EcoR Ⅰ和Hind Ⅲ酶切位点的引物，并通过引物的定点突变，利用PCR方法从猪肺炎支原体168F485株扩增到其黏附因子P97基因的抗原决定簇R1区。扩增产物经双酶切后，重组到表达载体pET-32a(+)中。重组质粒经酶切鉴定，PCR鉴定和测序，结果证实猪肺炎支原体黏附因子P97基因的抗原决定簇R1区定向插入了质粒pET-32a(+)，且阅读框架正确。将其转化宿主菌BL21(DE3)的感受态细胞，然后经过诱导条件(诱导温度、诱导剂用量和诱导时间)的优化，结果发现在37℃下，IPTG终浓度为0.2mmol／L，诱导4h进行表达，可得到了一分子量约265kDa的融合蛋白，表达量可达约23.3％。通过SDS-PAGE分离的表达产物和有机溶剂提纯的表达产物分别经Western Blot和ELISA检测证实具有抗原反应原性，从而为猪气喘病免疫学检测试剂盒和基因工程疫苗的研制奠定了基础。更多还原

【Abstract】 Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. Attachment to host tissue is essential for colonization by most mucosal pathogens, such as Mycoplasma hyopneumoniae. According to the reported sequence of P97 gene in strain 168, a pair of primers was designed and the Rl region of P97 gene in strain 168F485 was amplicated by PCR. In this case, two restriction sites(EcoR I , Hind III) were added to the primers (P1,P2) separately. The PCR product was inserted into expression plasmid pET-32a(+) after restriction digest. Then the recombinant plasmid was identified by endonuclease analysis, PCR ampliation and DNA sequencing. The report showed that the recombinant plasmid had right open reading frame. It was transformed into competent cells of BL21(DE3), then was induced to express by 0.2mmol/L IPTG at 37℃ for 4 hours .The recombinant protein was nearly 23.3% of the total products. It was proved that the recombinant protein had immune activity through Western blot and ELISA analysis. This provided the basis for the future preparation of Mycoplasma hyopneumoniae test kit and the new vaccine against Mycoplasma hyopneumoniae.更多还原