人类肠道病毒71型衣壳蛋白全基因成和原核表达分析
发布时间：2019-01-16 04:23
【摘要】：人类肠道病毒71型（Human enterovirus71，简称EV71）属于小RNA病毒科肠道病毒属，人类肠道病毒A，核酸为单股正链RNA。目前已知EV71的感染可以导致手足口病、无菌性脑膜炎、脑炎、疱疹性咽峡炎和脊髓灰质炎样的麻痹性疾病等多种与神经系统相关的疾病。手足口病在美国和欧洲处于小范围流行的阶段，但是在1975保加利亚和1978年匈牙利有过两次大疫情爆发和高死亡率。近年来，EV71病毒的流行在亚太地区呈上升趋势，包括马来西亚、新加坡、台湾、日本、韩国、越南和中国大陆。 肠道病毒EV71型有A、B（B1、B2、B3、B4、B5）、C（C1、C2、C3、C4、C5）三个基因型，共11个亚型，病毒的颗粒为二十面体立体对称的球形结构，无包膜，直径约24～30nm，核酸为含有大约7400个核苷酸的单股正链RNA。RNA中仅有一个开放阅读框（ORF），编码含2194个氨基酸的多聚蛋白，在其两侧为5′和3′非编码区（UTRs），与其他肠道RNA病毒一样，在3′末端有多聚腺苷酸（polyA）尾，而其5′末端共价结合有一个小分子量的蛋白（VPg）VP1，P2和VP3三个蛋白暴露在病毒外壳的表面，而VP4包埋在病毒外壳的内侧与病毒核心紧密连接，因而抗原决定簇基本上位于VP1-VP3上。 近年来，已经开发出不同类型的EV71疫苗，但目前普遍处于临床前开发阶段，包括灭活疫苗、DNA疫苗、减毒活疫苗、亚单位疫苗、表位多肽疫苗和病毒样颗粒疫苗。这些疫苗在动物模型中的研究表明中和抗体的产生起关键作用。本研究就目前研究中缺乏批准上市的抗原、抗体（酶标或化学发光）试剂，造成疫苗活性评价中中和抗体效价测定的不准确性和可比性，我们构建了VP1，VP0，VP3三种原核表达质粒，成功的表达了VP1，VP0蛋白，并对其免疫原性做了初步评价而且验证了VP1是抗原决定中心，为EV71诊断试剂盒和亚单位疫苗的研究奠定基础。 （一） EV71相关基因片段的体外合成 根据GenBank中录入的EV71/Fuyang.Anhui.CHN/19.08/6株全基因序列，翻译成经大肠杆菌密码子优化后的VP1，VP0,VP3的基因核苷酸序列，利用OE-PCR原理设计全基因体外合成的特异性引物并进行体外合成。合成的全长片段TA克隆至pMD18T载体上，经过序列测定和最后的修正合成，成功合成三个外壳蛋白的基因片段。 （二）VP1，VP0，VP3的原核表达和VP1的免疫原性分析。 分别以三个测序正确的含有基因片段的质粒为模板，用含酶切位点的引物PCR扩增出DNA产物后克隆到原核表达载体pET-32a，分别构建的三种原核表达质粒pET-32a-VP1，pET-32a-VP0，pET-32a-VP3将三种原核表达质粒转入E.coliBL21(DE3)，经过IPTG诱导表达和纯化，，进行动物免疫EV71-VP1蛋白的动物免疫，对其免疫原性进行分析。成功构建了三个原核表达质粒对进行原核表达并用Ni-NTA亲和层析法纯化目的融合蛋白。其中VP1，VP0表达量较高，VP3表达量不高。VP1免疫新西兰大白兔和BABL/C小鼠并制备抗血清。ELISA检测pET-32a-VP1和手足口病患儿的血清呈特异性结合反应，其IgG抗体滴度达1：64000，pET-32a-VP0和手足口病抗体滴度:1：32000，pET32a蛋白与仅有患儿的血清较弱结合。说明我们利用原核密码子体外基因合成表达表达纯化的蛋白很好的保留了很好的抗原性，同时也反映了VP1是抗原决定中心。为今后EV71诊断试剂盒和亚单位疫苗的研究提供参考。 （三）基孔肯雅病毒衣壳蛋白C和包膜蛋白E2的全基因合成和原核表达分析 利用重叠延伸PCR方法体外合成基孔肯雅病毒外壳蛋白C和包膜蛋白E2的全基因序列，并且构建外壳蛋白C和包膜蛋白E2的原核表达质粒。同时根据Expasy软件对E2跨膜疏水的预测，构建缺失疏水区的E2(1-350)突变体原核表达质粒。将测序正确的原核质粒转化至BL21(DE3)，经IPTG诱导表达，SDS-PAGE检测重组质粒的表达情况。经IPTG诱导表达后，SDS-PAGE结果显示缺失突变体pET21b-E2(1-350)融合蛋白表达量较pET21b-E2(1-404)有明显提高。提示E2蛋白跨膜疏水区（351-378aa）对该蛋白的原核表达具有重要影响。
[Abstract]:Human enterovirus 71 (EV71) belongs to the genus Enterovirus of the small RNA viridae, and the human enterovirus A and the nucleic acid are single-stranded positive-chain RNA. At present, it is known that the infection of the EV71 can lead to a variety of nervous system-related diseases such as hand-foot-mouth disease, aseptic meningitis, encephalitis, herpetic angina, and poliomyelitis-like paralysis. Hand-foot-mouth disease is in a small-scale epidemic in the United States and Europe, but in 1975 and 1978, there were two large outbreaks and high mortality rates in Hungary. In recent years, the prevalence of the EV71 virus is on the rise in the Asia-Pacific region, including Malaysia, Singapore, Taiwan, Japan, South Korea, Vietnam and China. The EV71 of the enterovirus type is A, B (B1, B2, B3, B4, B5), C (C1, C2, C3, C4, C5) three genotypes, 11 subtypes, the particle of the virus is the spherical structure of the icosahedral three-dimensional symmetry, the non-enveloped, the diameter is about 24 to 30n. m, the nucleic acid is a single-stranded positive strand RNA containing about 7400 nucleotides. There is only one open reading frame (ORF) in the RNA, which encodes a multimeric protein containing 2194 amino acids, with both 5 and 3 non-coding regions (UTRs) on both sides thereof, and one of the other intestinal RNA viruses. For example, polyadenosanoic acid (polyA) tail is present at the end of 3, and the 5-terminal end of which is covalently bound to a small molecular weight protein (VPg) VP1, P2 and VP3 three proteins are exposed to the surface of the viral envelope, while VP4 is embedded in the inner side of the viral envelope and is in close contact with the virus core and thus the antigenic determinant is located substantially in the VP1-VP3 In recent years, different types of EV71 vaccines have been developed, but are currently in the pre-clinical development phase, including live vaccines, DNA vaccines, attenuated live vaccines, subunit vaccines, epitope polypeptide vaccines, and virus-like The study of these vaccines in animal models shows that the production of neutralizing antibodies is off The key effect of this study is that the present study lacks the reagent of the antigen, antibody (enzyme or chemiluminescence) to be listed in the present study, which results in the inaccuracy and comparability of the antibody titer determination in the evaluation of the vaccine activity. We construct three prokaryotic expression plasmids of VP1, VP0 and VP3, which are successfully expressed. VP1, VP0 protein, and the immunogenicity of VP1 and VP0 were first evaluated, and VP1 was the antigen-determining center, and it was the study of the EV71 diagnostic kit and subunit vaccine. Base. (I) EV71-related gene chip The in vitro synthesis of the segment is based on the whole gene sequence of the EV71/ Fuyang, Anhua. CHN/ 19. 08/ 6 strain entered in the GenBank, and is translated into the gene core acid sequence of VP1, VP0 and VP3 after being optimized by the E. coli codon, and the specific primer for the in vitro synthesis of the whole gene is designed by the OE-PCR principle. The synthetic full-length fragment TA is cloned into the pMD18T vector, and the three shells are successfully synthesized through the sequence measurement and the final modified synthesis. Prokaryotic Expression and V of VP1, VPO, VP3 and the three prokaryotic expression plasmids pET-32a-VP1, pET-32a-VP0 and pET-32a-VP3 were respectively constructed to transfer three prokaryotic expression plasmids to E. co. coli BL21 (DE3), and expressed and purified by IPTG, and the animal immunity of the animal immune EV71-VP1 protein is carried out and the immunogenicity of the three prokaryotic expression plasmids is analyzed, and the three prokaryotic expression plasmids are successfully constructed for prokaryotic expression and are affinity with the Ni-NTA. The target fusion protein is purified by a chromatography method, wherein the expression amount of VP1 and VP0 High, VP3 expression is not high. VP1 immune New Zealand white rabbits and BAB The serum of pET-32a-VP1 and hand-foot-mouth disease was detected by ELISA. The concentration of IgG antibody was 1: 64000, the level of pET-32a-VPO and the degree of antibody drop of hand-foot-mouth disease were 1: 32000 and the protein of pET32a. Only the serum of the children is weak binding. The expression of the purified protein by using the in vitro gene synthesis of the prokaryote is very good, the antigenicity is good, VP1 is an antigen-determining center. For future EV71 diagnostic kits and subs Reference to the study of the unit vaccine. (iii) the base-hole Kenya virus capsid protein C and the envelope protein E 2. The whole-gene synthesis and the prokaryotic expression analysis use the overlapping extension PCR method to synthesize the shell egg of the kaphenya virus in vitro. The whole gene sequence of the white C and the envelope protein E2, and the shell protein is constructed the prokaryotic expression plasmid of the C and the envelope protein E2 is constructed, and the E2 (E2 (E2) of the missing hydrophobic region is constructed according to the prediction of the E2 cross-membrane hydrophobic according to the expression of the Expression software. 1-350) the prokaryotic expression plasmid of the mutant. The correct prokaryotic plasmid is transformed into the BL21 (DE3), and the expression is induced by IPTG, and the SDS-The expression of the recombinant plasmid was detected by PAGE. After the expression of IPTG, the expression of the fusion protein of the deleted mutant pET21b-E2 (1-350) was higher than that of the pET21b.-E2 (1-404) was significantly improved. It was suggested that E2 protein cross-membrane hydrophobic region (351-378aa)
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
本文编号：2409458
[Abstract]:Human enterovirus 71 (EV71) belongs to the genus Enterovirus of the small RNA viridae, and the human enterovirus A and the nucleic acid are single-stranded positive-chain RNA. At present, it is known that the infection of the EV71 can lead to a variety of nervous system-related diseases such as hand-foot-mouth disease, aseptic meningitis, encephalitis, herpetic angina, and poliomyelitis-like paralysis. Hand-foot-mouth disease is in a small-scale epidemic in the United States and Europe, but in 1975 and 1978, there were two large outbreaks and high mortality rates in Hungary. In recent years, the prevalence of the EV71 virus is on the rise in the Asia-Pacific region, including Malaysia, Singapore, Taiwan, Japan, South Korea, Vietnam and China. The EV71 of the enterovirus type is A, B (B1, B2, B3, B4, B5), C (C1, C2, C3, C4, C5) three genotypes, 11 subtypes, the particle of the virus is the spherical structure of the icosahedral three-dimensional symmetry, the non-enveloped, the diameter is about 24 to 30n. m, the nucleic acid is a single-stranded positive strand RNA containing about 7400 nucleotides. There is only one open reading frame (ORF) in the RNA, which encodes a multimeric protein containing 2194 amino acids, with both 5 and 3 non-coding regions (UTRs) on both sides thereof, and one of the other intestinal RNA viruses. For example, polyadenosanoic acid (polyA) tail is present at the end of 3, and the 5-terminal end of which is covalently bound to a small molecular weight protein (VPg) VP1, P2 and VP3 three proteins are exposed to the surface of the viral envelope, while VP4 is embedded in the inner side of the viral envelope and is in close contact with the virus core and thus the antigenic determinant is located substantially in the VP1-VP3 In recent years, different types of EV71 vaccines have been developed, but are currently in the pre-clinical development phase, including live vaccines, DNA vaccines, attenuated live vaccines, subunit vaccines, epitope polypeptide vaccines, and virus-like The study of these vaccines in animal models shows that the production of neutralizing antibodies is off The key effect of this study is that the present study lacks the reagent of the antigen, antibody (enzyme or chemiluminescence) to be listed in the present study, which results in the inaccuracy and comparability of the antibody titer determination in the evaluation of the vaccine activity. We construct three prokaryotic expression plasmids of VP1, VP0 and VP3, which are successfully expressed. VP1, VP0 protein, and the immunogenicity of VP1 and VP0 were first evaluated, and VP1 was the antigen-determining center, and it was the study of the EV71 diagnostic kit and subunit vaccine. Base. (I) EV71-related gene chip The in vitro synthesis of the segment is based on the whole gene sequence of the EV71/ Fuyang, Anhua. CHN/ 19. 08/ 6 strain entered in the GenBank, and is translated into the gene core acid sequence of VP1, VP0 and VP3 after being optimized by the E. coli codon, and the specific primer for the in vitro synthesis of the whole gene is designed by the OE-PCR principle. The synthetic full-length fragment TA is cloned into the pMD18T vector, and the three shells are successfully synthesized through the sequence measurement and the final modified synthesis. Prokaryotic Expression and V of VP1, VPO, VP3 and the three prokaryotic expression plasmids pET-32a-VP1, pET-32a-VP0 and pET-32a-VP3 were respectively constructed to transfer three prokaryotic expression plasmids to E. co. coli BL21 (DE3), and expressed and purified by IPTG, and the animal immunity of the animal immune EV71-VP1 protein is carried out and the immunogenicity of the three prokaryotic expression plasmids is analyzed, and the three prokaryotic expression plasmids are successfully constructed for prokaryotic expression and are affinity with the Ni-NTA. The target fusion protein is purified by a chromatography method, wherein the expression amount of VP1 and VP0 High, VP3 expression is not high. VP1 immune New Zealand white rabbits and BAB The serum of pET-32a-VP1 and hand-foot-mouth disease was detected by ELISA. The concentration of IgG antibody was 1: 64000, the level of pET-32a-VPO and the degree of antibody drop of hand-foot-mouth disease were 1: 32000 and the protein of pET32a. Only the serum of the children is weak binding. The expression of the purified protein by using the in vitro gene synthesis of the prokaryote is very good, the antigenicity is good, VP1 is an antigen-determining center. For future EV71 diagnostic kits and subs Reference to the study of the unit vaccine. (iii) the base-hole Kenya virus capsid protein C and the envelope protein E 2. The whole-gene synthesis and the prokaryotic expression analysis use the overlapping extension PCR method to synthesize the shell egg of the kaphenya virus in vitro. The whole gene sequence of the white C and the envelope protein E2, and the shell protein is constructed the prokaryotic expression plasmid of the C and the envelope protein E2 is constructed, and the E2 (E2 (E2) of the missing hydrophobic region is constructed according to the prediction of the E2 cross-membrane hydrophobic according to the expression of the Expression software. 1-350) the prokaryotic expression plasmid of the mutant. The correct prokaryotic plasmid is transformed into the BL21 (DE3), and the expression is induced by IPTG, and the SDS-The expression of the recombinant plasmid was detected by PAGE. After the expression of IPTG, the expression of the fusion protein of the deleted mutant pET21b-E2 (1-350) was higher than that of the pET21b.-E2 (1-404) was significantly improved. It was suggested that E2 protein cross-membrane hydrophobic region (351-378aa)
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373
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本文编号：2409458
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