问号状钩端螺旋体外膜蛋白Loa22的克隆、表达及对豚鼠的免疫保护作用研究

Cloning and Expression of Leptospiral interragans Outer Membrane Protein Loa22 and its Immunoprotectivity Among Guinea-pigs

目的构建问号状钩体强毒赖型56601株外膜蛋白Loa22的重组质粒,表达Loa22蛋白,研究该蛋白对豚鼠的保护作用。方法以问号状赖型钩体56601株基因组为模板扩增目的基因,将Loa22基因克隆至原核表达载体PQE-31,构建PQE31-Loa22重组体,将其转入大肠杆菌M15中诱导表达目的蛋白。于0、2、4周将蛋白及对照PBS分别经豚鼠腹股沟、腋下免疫豚鼠。每次剂量为蛋白50μg/只,末次免疫后2周,用培养3d的56601株钩体从腹腔攻击各免疫组豚鼠(1ml/只)。连续观察15d,观察各免疫组豚鼠发病情况,并取各免疫组豚鼠的肺、肝、肾做病理切片。分析蛋白Loa22对豚鼠的免疫保护作用。结果成功扩增出Loa22基因,构建原核重组质粒PQE31-Loa22。重组质粒能在大肠杆菌M15中高效表达Loa22蛋白。用Loa22蛋白主动免疫的豚鼠用56601株钩体攻击,结果显示无发病现象,而免疫对照组的豚鼠中出现了食欲不振、活动减少等现象,病理切片有明显改变。结论成功构建了Loa22重组原核表达质粒,表达纯化的蛋白可以使豚鼠抵抗同型钩体的攻击,免疫保护作用为82%。

Objective A prokaryotic expression plasmid PQE31 Loa22 containing the outer membrane protein Loa22 gene of Leptospira Interragans was constructed and its＇ immunological protective efficacy among guinea pigs were investigated. Methods Primers were designed by primer5.0 software to the Loa22 gene sequence of Leptospira interrogans reser- voir. Polymerase chain reaction （PCR） was used to amplify the Loa22 gene and Loa22 gene fragment was isolated. The fragment of Loa22 gene was directly ligated into prokaryotic expression vector PQE31 to construct the PQE31 - Loa22 plasmid. The recombinant plasmid was transfected into M15 and to be expressed. Guinea pigs with 150g body weight were immunized with Loa22 protein and/or PBS buffer intramuscularly at 2 - week interval for three times, and then challenged with L. interrogans, strain 56601. Results The Loa22 gene was successfully amplified and subcioned into prokaryotic expression vector PQE31. The expression of recombinant PQE31 - Loa22 could be expressed in M15. The guinea- pigs immunized with Loa22 protein were challenged with L. interrogans strain 56601, and they showed resistance to infection but the PBS - immunized ginea pigs not. Conclusions The Leptospira out membrane protein Loa22 gene is successfully amplified and subcloned into prokaryotic expression plasmid PQE31. The expressed and purified Loa22 protein could protect immunized guinea - pigs from homogenious Leptospira infection, and the protective rate is 82 %.