| 表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建 |
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| 引用本文： | 许崇波,卫广.表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建[J].生物工程学报,2002,18(2):216-220. |
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| 作者姓名： | 许崇波 卫广 |
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| 作者单位： | 1. 宁夏大学生物工程系,银川,750021
2. 辽宁省益康生物制品厂,辽阳,111000 |
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| 摘 要： | 利用PCR技术，从大肠杆菌C83902质粒中扩增出K88ac基因、ST1突变基因和LTB基因，通过分离、纯化、内切酶酶切、连接和转化，构建了含K88ac-ST1-LTB融合基因表达载体的重组菌株BL21（DE3）（pXKST3LT5)。经酶切鉴定和DNA序列分析证实，构建的重组质粒pXKST3LT5中含有K88ac-ST1-LTB融合基因，且基因序列和阅读框架均正确。经ELISA检测，重组菌株表达的K88ac-ST1-LTB融合蛋白能够被ST1单抗、LTB和K88ac抗体识别。经乳鼠灌胃试验证实，表达的融合蛋白已丧失天然ST1肠毒素的活性。免疫实验结果表明，K88ac-ST1-LTB融合蛋白能够诱发小白鼠产生抗体，该抗体具有中和天然ST1肠毒素的毒性作用，表明构建的重组菌株可以作为预防仔猪黄、白痢基因工程菌苗的候选菌株。
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| 关 键 词： | K88ac基因 ST1基因 LTB基因 融合基因 融合蛋白 基因表达 大肠杆菌 基因工程菌株 菌苗 黄白痢 |
| 文章编号： | 1000-3061(2002)02-0216-05 |
| 修稿时间： | 2001年8月22日 |
Construction of Recombinant Strain Expressing Enterotoxigenic Escherichia coli K88ac-ST1- LTB Fusion Protein |
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| XU Chong Bo,WEI Guang Sen.Construction of Recombinant Strain Expressing Enterotoxigenic Escherichia coli K88ac-ST1- LTB Fusion Protein[J].Chinese Journal of Biotechnology,2002,18(2):216-220. |
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| Authors: | XU Chong Bo WEI Guang Sen |
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| Affiliation: | Department of Biotechnology, Ningxia University, Yinchuan 750021, China. xcb921@sohu.com |
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| Abstract: | K88ac genes, heat stable enterotoxin I (ST 1) mutant genes and heat labile enterotoxin B subunit (LT B) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac ST 1 LT B fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac ST 1 LT B fusion protein was highly expressed in recombinant strain BL21(DE3)(pXKST3LT5) and the expression level of the K88ac ST 1 LT B fusion protein was about 75.53% of total cellular protein by SDS PAGE and thin layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to re cognize ST 1 \%in vitro.\% These sera antibodies were able to neutralize the biological activity of native ST 1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain. |
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| Keywords: | K88ac gene heat stable enterotoxin I gene heat labile enterotoxin B subunit gene fusion gene fusion protein gene expression |
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