猪胸膜肺炎放线杆菌溶血毒素的克隆表达及其对小鼠 免疫保护作用

Cloning and Expression of Hemolytic-toxin from Actinobacillus pleuropneu-moniae and the Immunoprotection in Mice

摘要:

将猪胸膜肺炎放线杆菌血清3型分离株的ApxⅡA、ApxⅢA、ApxⅣA基因和血清5型分离株的ApxⅠA基因分别克隆到原核表达载体pGEX-5X-3, 并在大肠杆菌BL21中进行表达。SDS-PAGE结果表明重组菌表达的最佳条件为诱导时间2小时和IPTG终浓度1mmol/L。通过硫酸铵盐析和Sephadex G-200凝胶过滤层析纯化表达产物。Western blot检测结果显示表达产物具有免疫活性。按照不同组合将表达产物与弗氏佐剂等比例混合, 制备3种亚单位疫苗。并在30日龄和45日龄免疫小白鼠, 在60日龄分别用血清1、3、5、7和10型胸膜肺炎放线杆菌攻毒。血清1、5和7型胸膜肺炎放线杆菌攻毒后, 3种亚单位疫苗分别提供58.4%、66.6%和91.7%的保护率。试验结果表明重组蛋白具有免疫保护作用, 且含有四种融合蛋白的亚单位疫苗免疫保护效果最佳。

Abstract:

The ApxⅡA、ApxⅢA、ApxⅣA genes from Actinobacillus pleuropneumoniae serotype 3 and the ApxⅠA gene from Actinobacillus pleuropneumoniae serotype 5 were respectively cloned into the prokaryotic expression vector pGEX-5X-3.Then the recombinant expression plasmids were respectively transformed into E.coli BL 21 and fusion pro-tein expression were induced by IPTG. The expression products were purified by precipitation with ammonium sulfate and chromatography on Sephadex G-200. SDS-PAGE indicated that the productsexpressed at a high level when the recombi-nant E.coli BL21 was induced 2h, joining IPTG to final concentration 1mmol/L. Western blot analysis showed that the expression products had immunogenicity and specificity.Subunit vaccines were made by different purified expression products and Freund's adjuvant. Mice were immunized at 30 days and 45 days with the subunit vaccines. Then the mice were challenged with the APP of serotype 1, 3, 5,7or 10 at 60 days. The result of animal immunoprotection test showed that subunit vaccines (ApxⅠA +ApxⅣA, ApxⅠA + ApxⅢA +ApxⅣA ,ApxⅠA + ApxⅡA + ApxⅢA + ApxⅣA) could offer 58.4%、66.6%、91.7% protection in mice against the challenge of serotype 1, 5 and 7 APP, respectively. These results suggested that the recombinant proteins had good immunogenicity and the subunit vaccine containing four kind of re-combinant proteins could induce better immunoprotection.