重组产气荚膜梭菌α毒素突变体的表达与免疫保护力评价

Expression and Protective Efficacy of Clostridium perfringens α toxin derivative

关键词:  A型产气荚膜梭菌CPA  突变  Th细胞表位  鞭毛蛋白  重组表达  毒力  抗原性

Key words:  Clostridium perfringens CPA  mutation  Th cell epitope  flagellin  recombinant expression  virulence  antigenicity

摘要:

本研究旨在获得产气荚膜梭菌α毒素(CPA)的重组突变体,并评价其毒力及免疫原性。对已知的产气荚膜梭菌CPA编码基因进行优化设计,同时引入包括第56位和第130位的天冬氨酸(D)突变为甘氨酸(G),及第275位的酪氨酸(Y)和第336位的天冬氨酸(D)突变为天冬酰胺(N)的4个氨基酸突变。此外,在该基因5’端添加Th细胞表位(T)和鞭毛蛋白(flagellin)N末端编码序列经人工合成获得基因片段GTFNCPAm4。将GTFNCPAm4克隆至原核表达载体pET-30a (＋)中进行表达与纯化,获得重组蛋白rTFNCPAm4。利用Western blot方法检测rTFNCPAm4与产气荚膜梭菌A型毒素抗血清的反应性,并采用小鼠检测纯化蛋白的毒力。随后,将rTFNCPAm4与Montanide ISA 201佐剂以1: 1(v/v)的比例混合乳化制备疫苗,免疫家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测兔血清的中和抗体效价。二免后21d,对家兔通过耳缘静脉注射1个家兔MLD剂量的A型产气荚膜梭菌毒素,以检测rTFNCPAm4对家兔的免疫保护效果。结果表明,rTFNCPAm4主要以非可溶的形式(包涵体)表达,且能与A型产气荚膜梭菌毒素抗血清反应；小鼠安全性实验显示,5.5mg/kg剂量的rTFNCPAm4对小鼠仍无致死性；免疫rTFNCPAm4后,每毫升的一免抗血清可中和10个小鼠MLD、二免抗血清可中和40个小鼠MLD的A型产气荚膜梭菌毒素；1个家兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了100%(4/4)的保护。以上结果表明,rTFNCPAm4具有良好的安全性和免疫原性,从而为A型产气荚膜梭菌基因工程亚单位疫苗的研制提供了重要的实验数据。

Abstract:

This study was conducted to obtain the Clostridium perfringens α toxin (CPA) derivative and to evaluate the virulence and immunogenicity of it. Based on the known sequence, four amino acid mutations, D56G, D130G, Y275N and D336N, were introduced into the gene of Clostridium perfringens CPA. Meanwhile, genes of Th cell and were added to 5’ of gene of CPA, respectively. Then the gene GTF_NCPA_m4 was optimized and synthesized and subsequently cloned into prokaryotic expression vector pET-30a (＋) for expression and purification to get recombinant protein rTF_NCPA_m4. Reactivity of the rTF_NCPA_m4with antiserum of Clostridium perfringens type A was detected by Western blot. Meanwhile, rTF_NCPA_m4was injected into mice to detect the virulence of it. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), four rabbits were immunized with rTF_NCPA_m4 emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer. The results showed that rTF_NCPA_m4 was presented predominantly in an insoluble form (inclusion bodies), and it could react with the antiserum of Clostridium perfringens type A. At the same time, rTF_NCPA_m4 with the injection volume of 5.5mg/kg was still not fatal to ICR mice. After the first immunization, sera from rabbits immunized with rTF_NCPA_m4 could neutralize 10 mice MLD Clostridium perfringens type A toxin per ml, and 40 mice MLD after twice immunization. Moreover, rabbits in rTF_NCPA_m4immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type A toxin challenge, whereas all of the rabbits died (4/4) in the control groups. These data suggest that rTF_NCPA_m4is a potential vaccine candidate for or genetic engineering subunit vaccine of Clostridium perfringens type A.