用于快速检测猪瘟抗体双功能分子的构建及表达
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摘要
猪瘟(Classical Swine Fever,CSF)是由猪瘟病毒引起的猪的一种急热性、致死性疾病。给世界养猪业造成了巨大的经济损失。对猪瘟抗体进行定期监测对猪瘟的防制具有重要意义,针对目前还没有一种适合基层单位使用的、简便、快速、稳定的猪瘟抗体检测方法的现状,本项目根据红细胞凝集试验的原理开展用于快速检测猪瘟抗体的双功能试剂方面的研究。研究主要结果如下:
1、双功能分子的拼接及其原核表达载体的构建
采用重叠延伸拼接(SOE)PCR的方法将2E8ScFv(抗人红细胞H抗原单链抗体基因)和mE2(猪瘟E2基因主要抗原区基因)拼接成融合双功能分子,命名为2E8mE2,将融合基因克隆入PMD18-T Simple载体,测序正确后,再亚克隆入PET-DsbA原核表达载体,通过PCR和酶切鉴定,均能获得大约1125bp的目的条带。表明原核表达载体PET-DsbA-2E8mE2构建成功。
2、2E8mE2重组蛋白的表达
将PET-DsbA-2E8mE2重组质粒转化BL21(DE3)PlysS原核表达菌株,用常规方法进行表达,经超声波裂解,SDS-PAGE电泳分析重组蛋白的性质,然后通过不同的温度、IPTG浓度、诱导时间的梯度来筛选最佳表达条件。结果表明:2E8mE2重组蛋白获得了高效表达,占细菌总量的30%;表达产物大部分是以不溶的包涵体形式存在;在37℃,IPTG浓度为0.3mmol/L,诱导时间为3h的条件下重组蛋白可获得最佳表达。
分别用6×His单克隆抗体和CSFV高免血清对表达的重组蛋白进行Western-blot检测,结果显示:重组蛋白与6×His单克隆抗体及CSFV阳性血清均呈阳性反应,呈现清晰的特异性反应条带,而空载体诱导菌的菌体蛋白无上述反应条带。表明所表达的重组蛋白是特异的。
3、2E8mE2重组蛋白包涵体的复性及检测
超声波破碎处理重组菌,收集重组蛋白包涵体,用0.5%TritonX-100和1mol/L NaCl洗涤包涵体后用尿素溶解,利用Ni-NTA树脂对溶解的蛋白进行亲和层析纯化,然后利用谷胱甘肽再氧化法对纯化的变性溶解蛋白进行复性,最后利用间接ELISA方法测定复性后的重组蛋白与CSFV高免血清的反应性,结果显示:用洗涤的方法初步纯化后,目的蛋白纯度已经能达到86%,再通过亲和层析法的进一步纯化,蛋白纯度高达92%,纯化后的重组蛋白用谷胱甘肽再氧化法进行复性,复性后的重组蛋白与CSFV高免血清的反应性明显高于复性前的变性蛋白,提高了约3-4倍,表明复性是成功的。
Classical swine fever(CSF) caused by Classical swine fever virus(CSFV) is a kind of urgent febricity and fatal disease. It has made a great loss in world's swine industry.monitoring regulaurly CSF antibody has important significance with control of CSF, at present, because there is still no a convenient and fast and stable detective method of CSF antibody to refer to primary level,the study of bifunctional reagent of detecting CSF antibody quickly performed according to the principle of haemagglutination.The main result of study as follow:
1. Splicing of bifunctional molecule and construction of prokaryotic vector
2E8ScFv (ScFv gene against H antigen of human erythrocyte) and mE2(main antigen region of E2 gene of CSF) were spliced by over lap extension(SOE) PCR and assembled bifunctional molecule named 2E8mE2, then it was cloned into PMD18-T Simple vector,the sequenced recombinant bifunctional molecule was sub-cloned into prokaryotic vector PET-DsbA and indentified by PCR and enzyme digest.Result showed that PCR and enzyme digest detected a 1125bp target band.The result indicated construction of prokaryotic vector PET-DsbA-2E8mE2 had been successful.
2.Expression of the 2E8mE2 recombinant protein
Recombinant vector PET-DsbA-2E8mE2 was transformed into the BL21 (DE3) PlysS E coli cell and expressed by common method, sonication of the bacterial cells and solubility analysis of recombinant protein by SDS-PAGE, then choice of expression conditions by grads of temperature and IPTG concentration and induce time.Result indicated the recombinant protein was gained high expression in the form of inclusion body at the level up to 30% total bacterial protein; the best temperature and IPTG concentration and induce time of recombinant protein expression were 37℃and 0.3mmol/L IPTG and 3h,respectively.
Detection of the recombinant protein with 6xHis monoclone antibody and positive serum against CSFV respectively by Western-blot, result showed that recombinant protein could react with 6xHis monoclone antibody and positive serum against CSFV respectively and showed limpid and specific reaction band which negative control didn't. The Result indicated expressed recombinant protein was specific.
3.Renaturation of inclusion body of the 2E8mE2 recombinant protein and detection of renatured recombinant protein
Sonication of the bacterial cells and collection of inclusion body of the recombinant protein, the inclusion body was washed with 0.5% TritonX-100 and 1mol/L NaCl and then subjected to denaturation in 8mol/L urea, then the denatured protein was purified with Ni-NTA resin by affinity chromatograph, at last the purified and denatured protein was renatured by glutathione reoxidation and detection of reactivity of the renatured recombinant protein and positive serum against CSFV by indirect ELISA. Result showed that the purity of interest protein reached 86% after washed and sublimation of purity of the interest protein reached 92% after purified with Ni-NTA resin by affinity chromatograph so that it may utend renature, reactivity of the renatured recombinant protein and positive serum against CSFV was boosted obviously contrasting to the denatured recombinant protein, result indicated renaturation of the recombinant protein was successful.
1、双功能分子的拼接及其原核表达载体的构建
采用重叠延伸拼接(SOE)PCR的方法将2E8ScFv(抗人红细胞H抗原单链抗体基因)和mE2(猪瘟E2基因主要抗原区基因)拼接成融合双功能分子,命名为2E8mE2,将融合基因克隆入PMD18-T Simple载体,测序正确后,再亚克隆入PET-DsbA原核表达载体,通过PCR和酶切鉴定,均能获得大约1125bp的目的条带。表明原核表达载体PET-DsbA-2E8mE2构建成功。
2、2E8mE2重组蛋白的表达
将PET-DsbA-2E8mE2重组质粒转化BL21(DE3)PlysS原核表达菌株,用常规方法进行表达,经超声波裂解,SDS-PAGE电泳分析重组蛋白的性质,然后通过不同的温度、IPTG浓度、诱导时间的梯度来筛选最佳表达条件。结果表明:2E8mE2重组蛋白获得了高效表达,占细菌总量的30%;表达产物大部分是以不溶的包涵体形式存在;在37℃,IPTG浓度为0.3mmol/L,诱导时间为3h的条件下重组蛋白可获得最佳表达。
分别用6×His单克隆抗体和CSFV高免血清对表达的重组蛋白进行Western-blot检测,结果显示:重组蛋白与6×His单克隆抗体及CSFV阳性血清均呈阳性反应,呈现清晰的特异性反应条带,而空载体诱导菌的菌体蛋白无上述反应条带。表明所表达的重组蛋白是特异的。
3、2E8mE2重组蛋白包涵体的复性及检测
超声波破碎处理重组菌,收集重组蛋白包涵体,用0.5%TritonX-100和1mol/L NaCl洗涤包涵体后用尿素溶解,利用Ni-NTA树脂对溶解的蛋白进行亲和层析纯化,然后利用谷胱甘肽再氧化法对纯化的变性溶解蛋白进行复性,最后利用间接ELISA方法测定复性后的重组蛋白与CSFV高免血清的反应性,结果显示:用洗涤的方法初步纯化后,目的蛋白纯度已经能达到86%,再通过亲和层析法的进一步纯化,蛋白纯度高达92%,纯化后的重组蛋白用谷胱甘肽再氧化法进行复性,复性后的重组蛋白与CSFV高免血清的反应性明显高于复性前的变性蛋白,提高了约3-4倍,表明复性是成功的。
Classical swine fever(CSF) caused by Classical swine fever virus(CSFV) is a kind of urgent febricity and fatal disease. It has made a great loss in world's swine industry.monitoring regulaurly CSF antibody has important significance with control of CSF, at present, because there is still no a convenient and fast and stable detective method of CSF antibody to refer to primary level,the study of bifunctional reagent of detecting CSF antibody quickly performed according to the principle of haemagglutination.The main result of study as follow:
1. Splicing of bifunctional molecule and construction of prokaryotic vector
2E8ScFv (ScFv gene against H antigen of human erythrocyte) and mE2(main antigen region of E2 gene of CSF) were spliced by over lap extension(SOE) PCR and assembled bifunctional molecule named 2E8mE2, then it was cloned into PMD18-T Simple vector,the sequenced recombinant bifunctional molecule was sub-cloned into prokaryotic vector PET-DsbA and indentified by PCR and enzyme digest.Result showed that PCR and enzyme digest detected a 1125bp target band.The result indicated construction of prokaryotic vector PET-DsbA-2E8mE2 had been successful.
2.Expression of the 2E8mE2 recombinant protein
Recombinant vector PET-DsbA-2E8mE2 was transformed into the BL21 (DE3) PlysS E coli cell and expressed by common method, sonication of the bacterial cells and solubility analysis of recombinant protein by SDS-PAGE, then choice of expression conditions by grads of temperature and IPTG concentration and induce time.Result indicated the recombinant protein was gained high expression in the form of inclusion body at the level up to 30% total bacterial protein; the best temperature and IPTG concentration and induce time of recombinant protein expression were 37℃and 0.3mmol/L IPTG and 3h,respectively.
Detection of the recombinant protein with 6xHis monoclone antibody and positive serum against CSFV respectively by Western-blot, result showed that recombinant protein could react with 6xHis monoclone antibody and positive serum against CSFV respectively and showed limpid and specific reaction band which negative control didn't. The Result indicated expressed recombinant protein was specific.
3.Renaturation of inclusion body of the 2E8mE2 recombinant protein and detection of renatured recombinant protein
Sonication of the bacterial cells and collection of inclusion body of the recombinant protein, the inclusion body was washed with 0.5% TritonX-100 and 1mol/L NaCl and then subjected to denaturation in 8mol/L urea, then the denatured protein was purified with Ni-NTA resin by affinity chromatograph, at last the purified and denatured protein was renatured by glutathione reoxidation and detection of reactivity of the renatured recombinant protein and positive serum against CSFV by indirect ELISA. Result showed that the purity of interest protein reached 86% after washed and sublimation of purity of the interest protein reached 92% after purified with Ni-NTA resin by affinity chromatograph so that it may utend renature, reactivity of the renatured recombinant protein and positive serum against CSFV was boosted obviously contrasting to the denatured recombinant protein, result indicated renaturation of the recombinant protein was successful.
引文
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