新城疫病毒强毒株F蛋白裂解位点区域基因的串联表达及其表达产物的应用研究
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摘要
本文利用E.coli原核表达系统对新城疫病毒(NDV)融合蛋白(F)裂解位点区域(fusion glycoprotein cleavage-activation site region，F_(CASR))基因进行了串联表达，并应用表达产物及其多肽抗体分别对NDV强弱毒株抗血清和NDV强弱毒株进行间接ELISA分析。该研究对新城疫病毒强弱毒株的鉴别诊断进行了有益的探索。主要研究内容如下：
1．人工设计，分段合成97bp的双链核苷酸序列(该序列编码产物含有NDV强毒株F蛋白裂解位点区域14个氨基酸的两个重复，中间通过两个疏水性氨基酸作为linker进行串联)，将合成的基因片段分别磷酸化，两两退火后，克隆入融合表达载体pET-32a(+)，并转化大肠杆菌BL21，用IPTG进行诱导表达，SDS-PAGE电泳可见约23.6kD大小的融合蛋白。将筛选到的重组阳性克隆命名为pET-F。经序列测定分析表明克隆基因与人工合成设计的基因序列一致。
2．将重组融合表达载体pET-F转化大肠杆菌BL21，挑取阳性克隆培养，通过摸索不同诱导时间、不同IPTG浓度，对融合蛋白的表达条件进行了优化，优化后的条件为：37℃振荡培养，待细菌生长至OD_(603)为0.4～0.6时(大约需2～3h)，加入IPTG至终浓度为0.1mM，30℃继续振荡培养2～3h。由于诱导温度较低并且所表达的融合蛋白分子量较小，所以融合蛋白以可溶性的形式存在。经螯和亲和层析纯化了该融合蛋白，为以后进一步研究提供了方便。另外，本研究将表达产物进行SDS-PAGE，然后回收目的条带制备抗原，并免疫家兔，制备抗血清，经Western-blotting检测结果显示抗体滴度较高。
3．利用纯化的目的蛋白对NDV强毒株F48E10和Ⅳ系疫苗株的抗血清进行间接ELISA检测，同时比较了OPD和TMB两种底物显色结果的差异。试验结果显示：F48E10强毒株和Ⅳ系疫苗株抗血清均有反应梯度，但Ⅳ系疫苗株抗血清反应比F48E10强毒株抗血清稍强；以OPD作底物时的OD值明显大于以TMB作底物时的OD值。另外，本研究通过兔抗NDV强毒株F_(CASR)多肽抗体与NDV强毒株F48E10和Ⅳ系疫苗株的间接ELISA分析，比较了该多肽抗体与NDV强弱毒株的反应性，结果反应性高低依次为：Ⅳ系疫苗株＞F48E10株＞F48E10株(0.1％胰酶处理)＞Ⅳ系疫苗株(0.1％胰酶处理)。
In this paper, the gene of fusion glycoprotein cleavage-activation site region of NDV virulent strain was repeated expressed in E.coli prokaryotic expression system. The expressed product was used to detect the antisera of virulent and avirulent strain of NDV by indirect ELISA. In addition, the antipeptide antibody of the fusion glycoprotein was used to detect the ivrulent and avirulent strain of NDV by indirect ELISA. This study indcludes:
1. In this study, the synthesized oligonucleotide (coding two same 14aa of fusion glycoprotein cleavage-activation site region of NDV virulent strain and two hydrophobic amino acids in between) was cloned into fusion-expression vector pET-32a(+) to construct the recombinant vector pET-F. The pET-F was then transformed into E.coli BL21 and the objective fusion protein was highly expressed by IPTG inducing. The expressed product of positive clone could be proved to be about 23.6kD by SDS-PAGE. The result of sequencing corresponded with what designed.
2. Recombinant fusion expression vector was transformed into E.coli strain BL21. The positive clone was selected to culture. The optimized expression conditions of the fusion protein was:30℃, 0.1 mM IPTG, incubating 2~3hrs after induction. For the molecular weight of the fusion protein is small and the temperature of culture is low, the expressed protein is soluble in E.coli. The result of SDS-PAGE showed that the protein was in supemate. The protein was purified by metal chelated chromatography. The purified protein will be utilized for further study. In addition, the expressed product which was reclaimed by SDS-PAGE was used to inoculate tame rabbit to raise antibodies. The result of Western-blot showed that the liter of the antibodies was rather high.
3. In the study, the purified protein was used to detect the anti-serum of virulent and avirulent strain of NDV by indirect ELISA. The aim is to detect their reactivity. In the same time, the results of the substrate of OPD and TMB were contrasted. The result showed that the reaction ladder of antisera of virulent and avirulent strain was obvious, and the OD data of antisera of avirulent strain were higher than the antisera of virulent strain; The OD data
using OPD as substrate were higher than using TMB as substrate. In addition, the antipeptide antibody of FCASR of NDV virulent strain was used to detect the virulent and avirulent strain of NDV by indirect ELISA. The reaction results (OD data) were: IV strain >F48E10 strain > F48E10 strain (treated with trypsin)> IV strain (treated with trypsin).
1．人工设计，分段合成97bp的双链核苷酸序列(该序列编码产物含有NDV强毒株F蛋白裂解位点区域14个氨基酸的两个重复，中间通过两个疏水性氨基酸作为linker进行串联)，将合成的基因片段分别磷酸化，两两退火后，克隆入融合表达载体pET-32a(+)，并转化大肠杆菌BL21，用IPTG进行诱导表达，SDS-PAGE电泳可见约23.6kD大小的融合蛋白。将筛选到的重组阳性克隆命名为pET-F。经序列测定分析表明克隆基因与人工合成设计的基因序列一致。
2．将重组融合表达载体pET-F转化大肠杆菌BL21，挑取阳性克隆培养，通过摸索不同诱导时间、不同IPTG浓度，对融合蛋白的表达条件进行了优化，优化后的条件为：37℃振荡培养，待细菌生长至OD_(603)为0.4～0.6时(大约需2～3h)，加入IPTG至终浓度为0.1mM，30℃继续振荡培养2～3h。由于诱导温度较低并且所表达的融合蛋白分子量较小，所以融合蛋白以可溶性的形式存在。经螯和亲和层析纯化了该融合蛋白，为以后进一步研究提供了方便。另外，本研究将表达产物进行SDS-PAGE，然后回收目的条带制备抗原，并免疫家兔，制备抗血清，经Western-blotting检测结果显示抗体滴度较高。
3．利用纯化的目的蛋白对NDV强毒株F48E10和Ⅳ系疫苗株的抗血清进行间接ELISA检测，同时比较了OPD和TMB两种底物显色结果的差异。试验结果显示：F48E10强毒株和Ⅳ系疫苗株抗血清均有反应梯度，但Ⅳ系疫苗株抗血清反应比F48E10强毒株抗血清稍强；以OPD作底物时的OD值明显大于以TMB作底物时的OD值。另外，本研究通过兔抗NDV强毒株F_(CASR)多肽抗体与NDV强毒株F48E10和Ⅳ系疫苗株的间接ELISA分析，比较了该多肽抗体与NDV强弱毒株的反应性，结果反应性高低依次为：Ⅳ系疫苗株＞F48E10株＞F48E10株(0.1％胰酶处理)＞Ⅳ系疫苗株(0.1％胰酶处理)。
In this paper, the gene of fusion glycoprotein cleavage-activation site region of NDV virulent strain was repeated expressed in E.coli prokaryotic expression system. The expressed product was used to detect the antisera of virulent and avirulent strain of NDV by indirect ELISA. In addition, the antipeptide antibody of the fusion glycoprotein was used to detect the ivrulent and avirulent strain of NDV by indirect ELISA. This study indcludes:
1. In this study, the synthesized oligonucleotide (coding two same 14aa of fusion glycoprotein cleavage-activation site region of NDV virulent strain and two hydrophobic amino acids in between) was cloned into fusion-expression vector pET-32a(+) to construct the recombinant vector pET-F. The pET-F was then transformed into E.coli BL21 and the objective fusion protein was highly expressed by IPTG inducing. The expressed product of positive clone could be proved to be about 23.6kD by SDS-PAGE. The result of sequencing corresponded with what designed.
2. Recombinant fusion expression vector was transformed into E.coli strain BL21. The positive clone was selected to culture. The optimized expression conditions of the fusion protein was:30℃, 0.1 mM IPTG, incubating 2~3hrs after induction. For the molecular weight of the fusion protein is small and the temperature of culture is low, the expressed protein is soluble in E.coli. The result of SDS-PAGE showed that the protein was in supemate. The protein was purified by metal chelated chromatography. The purified protein will be utilized for further study. In addition, the expressed product which was reclaimed by SDS-PAGE was used to inoculate tame rabbit to raise antibodies. The result of Western-blot showed that the liter of the antibodies was rather high.
3. In the study, the purified protein was used to detect the anti-serum of virulent and avirulent strain of NDV by indirect ELISA. The aim is to detect their reactivity. In the same time, the results of the substrate of OPD and TMB were contrasted. The result showed that the reaction ladder of antisera of virulent and avirulent strain was obvious, and the OD data of antisera of avirulent strain were higher than the antisera of virulent strain; The OD data
using OPD as substrate were higher than using TMB as substrate. In addition, the antipeptide antibody of FCASR of NDV virulent strain was used to detect the virulent and avirulent strain of NDV by indirect ELISA. The reaction results (OD data) were: IV strain >F48E10 strain > F48E10 strain (treated with trypsin)> IV strain (treated with trypsin).
引文
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