猪源产肠毒素性大肠杆菌K88、K99，987P、F41双基因串联表达的研究
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摘要
产肠毒素性大肠杆菌(ETEC)是引起仔猪腹泻的主要病原菌,黏附素在ETEC的致病过程中起着重要作用。从动物ETEC中发现的粘附素抗原有K88、K99、987P、F41、F42、F165、F17和F18等。而猪源粘附素抗原则以K88、K99、987P、F41最为常见。本研究实现了上述四种常见黏附素抗原的克隆表达,并在此基础上对K88-K99、987P-F41进行了串联表达。对表达蛋白进行了western-blot鉴定及反向间接血凝试验,结果表明所表达的融合蛋白具有良好的反应原性和免疫活性。为基因工程二价苗及多价苗的研制奠定了基础。研究内容如下:
1.利用PCR技术,以大肠杆菌C83907、C83644、C83710的质粒和C83707的基因组DNA为模板分别扩增不含信号肽的K88、K99、987P和F41基因。将其分别克隆到pMD18-t载体上,酶切鉴定成功构建重组质粒pMD18-K88、pMD18-K99、pMD18-987P、pMD18-F41。将其与pET30a载体分别经双酶切,将回收的基因小片段与载体大片段连接,得到的四种重组质粒。经酶切及PCR鉴定,并进行核苷酸序列分析,阳性质粒命名为pETK88、pETK99、pET987P、pETF41。将此四种重组质粒转化大肠杆菌BL21感受态细胞,IPTG诱导表达,经SDS-PAGE分析,目的基因得到高效表达。
2.在成功克隆K99基因的基础上,通过引物设计软件设计一对引物使扩增出的K88基因不含终止密码子。利用DNA重组技术将此K88基因亚克隆于pET30a表达载体中,得到阳性重组质粒命名为pETK88-2。进一步将K99基因融合到K88-2基因的下游,构建K88-K99串联基因,并实现其在大肠杆菌BL21中的高效表达。同时,对于F41-987P双基因串联表达,基本实验方法同K88-K99。区别在于,首先PCR扩增不含终止密码子的987P基因,将其克隆于pMD 18-T载体中,构建重组质粒,命名为pMD 18-987P-2。将987P-2基因插入到pETF41重组质粒的F41基因的上游。构建987P-F41串联基因,并实现其在大肠杆菌中的表达。
3. Western-blotting检测,结果表明,K88-K99、F41-987P串联表达蛋白能被大肠杆菌K88、K99、987P标准血清及F41抗血清识别。具有良好的反应原性。反向间接血凝实验结果表明,K88效价为2~8～2~(10) ,K99效价为2~7～2~8,F41效价为2~6～2~8 ,987P效价为2~8～2~(10),该串联表达蛋白具有良好的抗原性。表明构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基因工程疫苗的候选菌株。
The EnterotoxigenicEscherichia coli(ETEC) is an important pathogenic bacteria causing Colibacillus diarrhea of piglet. Adhesions gene plays an important role in the pathogenesis of disease.There are serveral kinds adhesions for example K88、K99、987P、F41、F42、F165、F17and F18 ,especially K88、K99、987P、F41 are more important.Both K88-K99 and 987P-F41 obtained co-expression on the base of single- expressed the four adhesions.It indicated that co-expression fusion-proteins had good reactivity and immunity activity.It established the foundation to manufacturing the gene engineering vaccine of preventing diarrhea of piglet.The results are as follows:
1. The DNA fragments of K88 \ K99\987P\F41 without signal peptide sequence were amplified by PCR from the plasmid DNA of E.coli C83907 \C83644\ C83710 and from the DNA of E.coli C83707. Then these amplified genes were inserted respectively into pMD18-t, obtained the recombinant plasmid pMD18-K88\ pMD18-K99\ pMD18-987P\ pMD18-F41 by identification. Then, the four recombinant plasmids and pET30a expression-carrier were digested separately by restriction endonuclease, were linked with T4 DNA Ligase, were recombined to be pET K88\ pET K99\ pET 987P\ pET F41. The analysis of restriction endonuclease-digesting and PCR and nucleotide sequence analysis confirmed that the fusion genes were constructed successfully. The result of SDS-PAGE indicated that the four target genes contained expression
2.The K88 gene which had no termination cipher was amplified on the base of cloning K99 gene . The k99 gene was set to the K88 gene downstream end by DNA recombined technology.The recombinant plasmid pET K88-K99 was constructed.The same to F41-987P.The difference was that the 987P gene which had no termination cipher was amplified .It was called 987p-2.The 987p-2 gene was set to the upstream end of F41 by DNA recombined technology. The recombinant plasmid pET 987P-F41 was constructed on the base.
3.The result of western-blotting indicated that the co-expression protein could be recognized by the antiserum against K88 and K99 and 987P and F41. By the reverse indirect hemagglutination assay,K88 potency was 2~8～2~(10) and K99 potency was 2~7～2~8 and F41 potency was 2~6～2~8 and 987P potency was 2~8～2~(10) .It indicated that the co-expression protein had the favourable antigenicity.It indicated that the constructed recombinant strain may be taken as the candidate strain to prevent the colibacillus diarrhea of piglet with Escherichia coli gene engineering vaccine.
1.利用PCR技术,以大肠杆菌C83907、C83644、C83710的质粒和C83707的基因组DNA为模板分别扩增不含信号肽的K88、K99、987P和F41基因。将其分别克隆到pMD18-t载体上,酶切鉴定成功构建重组质粒pMD18-K88、pMD18-K99、pMD18-987P、pMD18-F41。将其与pET30a载体分别经双酶切,将回收的基因小片段与载体大片段连接,得到的四种重组质粒。经酶切及PCR鉴定,并进行核苷酸序列分析,阳性质粒命名为pETK88、pETK99、pET987P、pETF41。将此四种重组质粒转化大肠杆菌BL21感受态细胞,IPTG诱导表达,经SDS-PAGE分析,目的基因得到高效表达。
2.在成功克隆K99基因的基础上,通过引物设计软件设计一对引物使扩增出的K88基因不含终止密码子。利用DNA重组技术将此K88基因亚克隆于pET30a表达载体中,得到阳性重组质粒命名为pETK88-2。进一步将K99基因融合到K88-2基因的下游,构建K88-K99串联基因,并实现其在大肠杆菌BL21中的高效表达。同时,对于F41-987P双基因串联表达,基本实验方法同K88-K99。区别在于,首先PCR扩增不含终止密码子的987P基因,将其克隆于pMD 18-T载体中,构建重组质粒,命名为pMD 18-987P-2。将987P-2基因插入到pETF41重组质粒的F41基因的上游。构建987P-F41串联基因,并实现其在大肠杆菌中的表达。
3. Western-blotting检测,结果表明,K88-K99、F41-987P串联表达蛋白能被大肠杆菌K88、K99、987P标准血清及F41抗血清识别。具有良好的反应原性。反向间接血凝实验结果表明,K88效价为2~8～2~(10) ,K99效价为2~7～2~8,F41效价为2~6～2~8 ,987P效价为2~8～2~(10),该串联表达蛋白具有良好的抗原性。表明构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基因工程疫苗的候选菌株。
The EnterotoxigenicEscherichia coli(ETEC) is an important pathogenic bacteria causing Colibacillus diarrhea of piglet. Adhesions gene plays an important role in the pathogenesis of disease.There are serveral kinds adhesions for example K88、K99、987P、F41、F42、F165、F17and F18 ,especially K88、K99、987P、F41 are more important.Both K88-K99 and 987P-F41 obtained co-expression on the base of single- expressed the four adhesions.It indicated that co-expression fusion-proteins had good reactivity and immunity activity.It established the foundation to manufacturing the gene engineering vaccine of preventing diarrhea of piglet.The results are as follows:
1. The DNA fragments of K88 \ K99\987P\F41 without signal peptide sequence were amplified by PCR from the plasmid DNA of E.coli C83907 \C83644\ C83710 and from the DNA of E.coli C83707. Then these amplified genes were inserted respectively into pMD18-t, obtained the recombinant plasmid pMD18-K88\ pMD18-K99\ pMD18-987P\ pMD18-F41 by identification. Then, the four recombinant plasmids and pET30a expression-carrier were digested separately by restriction endonuclease, were linked with T4 DNA Ligase, were recombined to be pET K88\ pET K99\ pET 987P\ pET F41. The analysis of restriction endonuclease-digesting and PCR and nucleotide sequence analysis confirmed that the fusion genes were constructed successfully. The result of SDS-PAGE indicated that the four target genes contained expression
2.The K88 gene which had no termination cipher was amplified on the base of cloning K99 gene . The k99 gene was set to the K88 gene downstream end by DNA recombined technology.The recombinant plasmid pET K88-K99 was constructed.The same to F41-987P.The difference was that the 987P gene which had no termination cipher was amplified .It was called 987p-2.The 987p-2 gene was set to the upstream end of F41 by DNA recombined technology. The recombinant plasmid pET 987P-F41 was constructed on the base.
3.The result of western-blotting indicated that the co-expression protein could be recognized by the antiserum against K88 and K99 and 987P and F41. By the reverse indirect hemagglutination assay,K88 potency was 2~8～2~(10) and K99 potency was 2~7～2~8 and F41 potency was 2~6～2~8 and 987P potency was 2~8～2~(10) .It indicated that the co-expression protein had the favourable antigenicity.It indicated that the constructed recombinant strain may be taken as the candidate strain to prevent the colibacillus diarrhea of piglet with Escherichia coli gene engineering vaccine.
引文
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