| 猪水肿毒素A亚基基因表达产物的生物学特性及抗体ELISA检测方法研究 |
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| 引用本文： | 赵战勤,张建民,卢顺,何华,吴斌,金梅林,陈焕春.猪水肿毒素A亚基基因表达产物的生物学特性及抗体ELISA检测方法研究[J].农业生物技术学报,2007,15(3). |
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| 作者姓名： | 赵战勤 张建民 卢顺 何华 吴斌 金梅林 陈焕春 |
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| 作者单位： | 华中农业大学动物医学院，农业微生物学国家重点实验室 |
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| 摘 要： | 通过TMpred生物学软件分析猪水肿毒素(SLT-IIe) A亚基蛋白质结构并设计引物、构建表达质粒使基因片段获得可溶性表达。SDS-PAGE和Western-blotting分析表明，表达产物具有可溶性从而主要存在于裂解上清液，易于纯化且具有良好免疫学活性。生物学毒性试验表明该可溶性蛋白不能致死小白鼠，也不具有Vero细胞毒性。体外细胞毒性中和试验表明表达产物的兔抗血清能高效中和SLT-IIe（中和度90.4℅）。在小鼠免疫保护力试验中，表达产物作为免疫原不能提供任何保护力。以表达产物为抗原建立的检测SLT-IIe抗体的间接ELISA方法具有良好的特异性、敏感性和稳定性，可用于临床猪水肿病的诊断。
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| 关 键 词： | 猪水肿病 SLT-IIe 生物学特性 ELISA |
| 收稿时间： | 2006-10-9 |
| 修稿时间： | 2007-3-5 |
Studying on Bio-characteristics of Expression Product of Shiga-like Toxin IIe A Subunit gene and ELISA Test |
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| Abstract: | In order to get the soluble expression products, Shiga-like toxin IIe A subunit was analyzed by TMpred software. Then a pair of primers were designed and the expression plasmid pGEX-A was constructed before being transformed into BL21 (DE3). SDS-PAGE and Western blotting results showed the gene fragment was expressed abundant soluble protein and the expression products had immunogenicity. The assay of it’s bio-characteristic toxicity showed that the soluble fusion protein lost the toxicity as SLT-IIe. Polyclonal antiserum produced by immunizing the New Zealand rabbits with the purified expression product restrained the toxicity of SLT-IIe to Vero E6 cells because it had significantly high neutralizing activity (90.4℅) against SLT-IIe. But the protective efficacy test showed that the immunization with the expression products could not reduce mortality of the mice. The ELISA method was established using the expressed protein to detect anti-SLT-IIe A IgG. The assay had excellent specificity, sensitivity and reduplication and could be applied to detect the edema disease in piglets. |
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