| 非洲猪瘟病毒VP73结构蛋白表达及鉴定 |
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| 引用本文： | 董 林,王艳萍,张春玲,沈志强.非洲猪瘟病毒VP73结构蛋白表达及鉴定[J].家畜生态学报,2013,34(4):62-65. |
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| 作者姓名： | 董 林 王艳萍 张春玲 沈志强 |
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| 作者单位： | 1.山东省滨州畜牧兽医研究院，山东 滨州 256600；2.山东绿都生物科技有限公司，山东 滨州 256600 |
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| 基金项目： | 山东省自然科学基金资助项目(ZR2010CQ012) |
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| 摘 要： |
为获得可作为血清学诊断抗原的体外诱导表达ASFV VP73 结构蛋白，根据GenBank登录的ASFV 基因序列，人工合成VP73主要抗原表位区基因克隆至pUC57载体中，设计1对引物，PCR扩增获得429 nt的VP73基因片段；将VP73基因片段使用EcoRI和SalI酶切后亚克隆至表达载体pGEX KG，经PCR、酶切、测序鉴定挑选阳性克隆，转化进表达菌株BL21(DM3)中进行体外诱导表达，SDS PAGE和Western blot分析蛋白表达及其活性。结果显示，成功构建表达重组载体pGEX KG VP73，且诱导有效表达了41Ku重组蛋白，表达量约占菌体总蛋白的35％；Western blot分析表达该蛋白具有良好的表达活性。试验为建立无感染性、快速、敏感的血清学诊断方法奠定了一定基础。
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| 关 键 词： | 非洲猪瘟病毒 VP73结构蛋白 表达及鉴定 |
Protein Expression and Identification of Structural VP73 for African Swine Fever Virus |
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| DONG Lin,WANG Yan-ping,ZHANG Chun-ling,SHEN Zhi-qiang.Protein Expression and Identification of Structural VP73 for African Swine Fever Virus[J].Acta Ecologae Animalis Domastici,2013,34(4):62-65. |
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| Authors: | DONG Lin WANG Yan-ping ZHANG Chun-ling SHEN Zhi-qiang |
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| Institution: | 1.Binzhou Animal Science and Veterinary Medicine Institute, Binzhou ,Shandong 256600,China;
2.Shandong Lvdu Bio Sciences & Technology Co.Ltd, Binzhou ,Shandong 256600,China |
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| Abstract: | The experiment was to obtain the ASFV structural protein VP73 that was expressed in vitro, which was as a diagnostic antigen. According to the gene sequence of ASFV VP73 in the gene bank, the major antigenic epitope gene of VP73 was synthesized and cloned into pUC57 vector. The VP73 gene of African Swine Fever Virus was amplified from DNA by PCR, the product of which was a 429 bp DNA segment. The purified VP73 gene was subcloned into pGEX KG vector by EcoRI and Sal enzymes digestion. The recombinant plasmid was identified by PCR and enzymes digestion. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP73 gene. The recombinant plasmid was transformed into BL21 (DM3).The recombinant protein was induced expression by IPTG which was analyzed by SDS PAEG and Western blot. The results show the recombinant plasmid was successfully built. The results of SDS PAGE revealed that the VP73 protein was expressed in pGEX KG vector. The optimal amount of the expressed fusion protein was 35% of total bacterial protein. It effectively expressed 41Ku recombinant protein and the Western blot analysis indicated a better immunologically reactive activity. The experiment has laid a foundation for the establishment of a rapid, sensitive serologically diagnostic method which is free of infectivity. |
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| Keywords: | ASFV VP73 structural protein expression and identification |
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