肠道病毒EV71 VP1基因的优化和免疫原性的研究

发布时间：2018-06-23 11:07

本文选题：肠道病毒EV71 + VP1基因　；参考：《南京医科大学》2011年博士论文

【摘要】：第一部分肠道病毒EV71衣壳蛋白VP1基因的优化目的:为了使肠道病毒EV71的主要中和抗原VP1基因能够在哺乳动物细胞内有效表达,按照哺乳动物细胞密码子使用的偏好,对VP1基因序列进行密码子优化(codon optimization, opt),并合成优化的VP1基因。方法:选取EV71 BJ4211株VP1基因(Genbank序列号EU024958.1)序列,用基因分析软件MacVector 7.2分析其基因编码序列,找出野生型VP1基因密码子使用的偏好性,并比较野生型EV71/BJ VP1的基因序列中与哺乳动物细胞密码子使用偏好不同的位点。对于与哺乳动物细胞使用偏好相同的密码子,用哺乳动物偏好的密码子替代野生型EV71/BJ VP1基因中使用偏好不同的密码子位点,然后设计出密码子优化的VP1基因。序列的优化还包括对基因mRNA的调整以使其更稳定和更易于在真核细胞中进行转录和翻译。密码子优化的VP1基因由德国Geneart公司合成,为便于目的基因的鉴定和插入,在基因两端分别加上限制性内切酶位点PstⅠ和BamHⅠ,装入骨架质粒pGA4,构建重组质粒pGA4/VP1-opt。将pGA4/VP1-opt转化大肠杆菌HB101,挑取单克隆,小量提取质粒鉴定;阳性克隆三次划氨苄青霉素(Amp)LB平板,获得含有pGA4/VP1-opt质粒的HB101菌种,20%甘油,-70℃保存。结果与结论:经酶切证实插入到载体pGA4中的基因片段大小正确,获得密码子优化的VP1基因。通过软件分析发现,与野生型VP1基因相比,密码子优化的VP1基因中哺乳动物细胞偏好的密码子出现频率增加,从而使其更适于在哺乳动物细胞中进行蛋白表达,但是其编码VP1蛋白的氨基酸序列不变,以保证原有的抗原构象。目的:为了提高EV71衣壳蛋白VP1基因在真核细胞的的表达和分泌水平,增强其免疫原性,以DNA疫苗为工具,通过选择合适的载体,改造基因表达和分泌的调控元件,设计并构建多种形式的密码子优化的VP1 DNA疫苗。通过分析不同形式的VP1 DNA疫苗在真核细胞的表达和分泌水平,及其免疫动物刺激产生抗体的能力,比较、评价何种形式的VP1蛋白表达和分泌最佳,具有良好的免疫原性,为筛选有效的EV71VP1蛋白候选疫苗奠定基础。方法:将pGA4/VP1-opt质粒用PstⅠ和BamHⅠ限制性内切酶进行双酶切,通过割胶纯化获得密码子优化的VP1基因,插入到真核表达载体pJW4303中,构建基因优化的VP1 DNA疫苗pJW4303/VP1,由于本研究后续的设计均是基于此优化的VP1基因,因此这种单纯的基因优化的VP1 DNA疫苗称之为“野生型(widetype,即VP1-wt)”。通过设计PCR引物P1和P2,以pGA4/VP1-opt质粒为模板,扩增获得含有NheⅠ和BamHⅠ酶切位点的VP1基因,克隆到pJW4303载体的tPA信号肽序列下游,构建含有tPA信号肽的VP1 DNA疫苗pJW4303/tPA-VP1(tPA-VP1),以促进蛋白的表达与分泌。为模拟自然状态下VP1易于自联形成二聚体的特点,设计双聚体VP1 DNA疫苗:合成PCR引物P3和P4,用P1和P4引物PCR扩增获得VP1插入片段1(Insert1,含NheⅠ和KpnⅠ酶切位点),用P3和P2引物PCR扩增获得VP1插入片段2(Insert2,含KpnⅠ和BamHⅠ酶切位点),将Insert1和Insert2同时克隆到pJW4303载体的tPA信号肽序列下游,构建含有tPA信号肽的双聚体VP1 DNA疫苗pJW4303/tPA-VP1-dimer(dimer)。由于免疫球蛋白IgG Fc(Fcγ)片段在机体免疫中的巨大优势,分别设计引物,PCR扩增含有KpnⅠ和BamHⅠ酶切位点的人Fcγ(huFcγ)和小鼠Fcγ(mFcγ)基因,将VP1-dimer质粒中的Insert2切掉,插入Fcγ基因,构建含Fcγ基因的VP1 DNA疫苗pJW4303/tPA-VP1-Fcγ(VP1- Fcγ),达到增强抗原与免疫细胞的接触,提高免疫原性的目的。构建的重组质粒转化大肠杆菌HB101,挑取单克隆,进行质粒提取、酶切和测序鉴定。鉴定正确的阳性克隆三次划平板,进行甘油菌种保存。鉴定成功的重组质粒进行质粒大量提取,转染293T细胞,收获转染上清和细胞裂解产物,通过酶联免疫标记法(ELISA)、间接免疫荧光分析(IFA)和蛋白质免疫印迹(Western blot)检测转染细胞中VP1蛋白的表达。提取的质粒还用于免疫动物,免疫方法采用肌肉注射加电转录活体基因导入方式,免疫剂量为200μg/兔。免疫在0、2、4和第8周进行,共4次。每次免疫前采血,分离血清,通过检测血清中特异性IgG抗体的应答水平,比较不同形式VP1 DNA疫苗的刺激产生抗体的能力,综合评价疫苗的免疫原性。结果和结论:基于密码子优化的VP1基因,成功设计和构建了五种形式的VP1 DNA疫苗:VP1-wt、tPA-VP1、VP1-dimer、VP1- huFcγ和VP1- mFcγ。五种形式的VP1 DNA疫苗均能在真核293T细胞中表达,加tPA信号肽后,VP1蛋白表达和细胞外分泌水平均明显增加;VP1-dimer的表达和分泌水平则进一步提高。VP1-huFc重组质粒不仅表达水平显著增加,其在Fc的引导下,细胞外分泌水平也大大提高。而加有mFcγ片段的VP1 DNA疫苗的表达和分泌则并不理想。五种DNA疫苗免疫新西兰白兔发现,tPA-VP1免疫原性也较好,产生抗体水平最高。
[Abstract]:Part one optimization of enterovirus EV71 capsid protein VP1 gene
Objective: in order to effectively express the main Neutralizing Antigen VP1 gene of enterovirus EV71 in mammalian cells, in accordance with the preference used by the codon of mammalian cells, the VP1 gene sequence was optimized by codon optimization (OPT), and the optimized VP1 gene was synthesized.
Methods: the VP1 gene (Genbank sequence number EU024958.1) sequence of EV71 BJ4211 strain was selected, and the gene coding sequence was analyzed with the gene analysis software MacVector 7.2. The preference of the wild type VP1 gene codon used was found, and the loci of the wild type EV71/BJ VP1 gene sequence with the mammalian codon were compared. Mammalian cells use the same codons with the same preference, and use the mammalian preferred codon instead of the wild type EV71/BJ VP1 gene to use different codon loci, and then design the codon optimized VP1 gene. The sequence optimization also includes the adjustment of the gene mRNA to make it more stable and easier to carry out in the eukaryotic cells. Transcriptional and translation. Codon optimized VP1 gene was synthesized by German Geneart company. In order to facilitate the identification and insertion of the target gene, the restriction endonuclease Pst I and BamH I were added at both ends of the gene. The recombinant plasmid pGA4/VP1-opt. was loaded into the skeleton plasmid pGA4, and the recombinant plasmid pGA4/VP1-opt. was constructed to transform pGA4/ VP1-opt into the Escherichia coli HB101, and to pick out the monoclonal and small amount. Plasmid identification was carried out; positive clones were distributed three times with ampicillin (Amp) LB plate, and HB101 strain containing pGA4/VP1-opt plasmid was obtained, and 20% glycerol was stored at -70 C.
Results and conclusion: the gene fragment inserted into the carrier pGA4 was proved to be correct in size, and the VP1 gene optimized by codon was obtained. By software analysis, it was found that the frequency of the mammalian preferred codons in the VP1 gene was increased in the VP1 gene optimized by the codon VP1, thus making it more suitable for the mammalian cells. The protein was expressed, but its amino acid sequence encoding VP1 protein remained unchanged to ensure the original antigen conformation.
Objective: in order to improve the expression and secretion of EV71 capsid protein VP1 gene in eukaryotic cells and enhance its immunogenicity, DNA vaccine was used as a tool to modify the regulatory elements of gene expression and secretion by selecting the appropriate carrier, and to design and construct a variety of forms of VP1 DNA vaccine optimized by codon. By analyzing different forms of VP1 DNA In the expression and secretion level of eukaryotic cells and the ability of the immune animals to stimulate the production of antibodies, the vaccine is compared to evaluate the best expression and secretion of VP1 protein. It has good immunogenicity, which lays the foundation for the screening of effective EV71VP1 protein vaccine.
Methods: the pGA4/VP1-opt plasmid was cut by Pst I and BamH I restriction endonuclease, and the VP1 gene optimized by codon was obtained by gapping and purified, and inserted into the eukaryotic expression vector pJW4303 to construct the VP1 DNA vaccine pJW4303/VP1, which was optimized by gene. The gene optimized VP1 DNA vaccine is called "widetype (VP1-wt)". Through the design of PCR primers P1 and P2, pGA4/VP1-opt plasmids are used as templates to amplify the VP1 gene containing Nhe I and BamH I, and clone into the peptide sequence of the pJW4303 carrier. 1 (tPA-VP1), in order to promote the expression and secretion of protein, designed the VP1 DNA vaccine of VP1: PCR primers P3 and P4, P1 and P4 primer PCR amplification by P1 and P4 primer PCR. Sert2, Kpn I and BamH I enzyme cutting site), Insert1 and Insert2 were cloned at the same time of the tPA signal peptide sequence of pJW4303 carrier, and the oligomer VP1 DNA vaccine containing tPA signal peptide was constructed. The human Fc gamma (huFc gamma) and the Fc gamma (mFc gamma) gene of the Kpn I and BamH I sites were cut off and inserted into the Fc gamma gene to construct VP1 DNA vaccine containing Fc gamma gene, which could enhance the contact between the antigen and immune cells and improve the immunogenicity. The recombinant plasmid was transformed into a large transformation plasmid. Enterobacteriaceae HB101 was selected to extract the monoclonal antibodies. The plasmid was extracted, identified by restriction enzyme digestion and sequencing. The correct positive clones were identified for the three time, and the glycerol bacteria were preserved.
The recombinant plasmid was successfully extracted, transfected into 293T cells, and harvested the transfected supernatant and cell lysis products. The expression of VP1 protein in transfected cells was detected by indirect immunofluorescence analysis (ELISA), indirect immunofluorescence analysis (IFA) and protein immunoblotting (Western blot). The extracted plasmid was also used in immune animal and immune prescription. The immunization dose was 200 u g/ rabbits. The immunization was carried out in 0,2,4 and eighth weeks. The immunization was carried out for 4 times. Before each immunization, blood was collected and serum was separated. By detecting the response level of the specific IgG antibody in the serum, the ability to produce antibodies with different forms of VP1 DNA vaccine was compared and the vaccine was evaluated in a comprehensive way. Phytophthora.
Results and conclusions: five types of VP1 DNA vaccines were successfully designed and constructed based on the codon optimized VP1 gene: VP1-wt, tPA-VP1, VP1-dimer, VP1- huFc gamma and VP1- mFc gamma.
Five forms of VP1 DNA vaccine could be expressed in eukaryotic 293T cells. After adding tPA signal peptide, the expression of VP1 protein and the level of exocrine secretion increased obviously, and the expression and secretion level of VP1-dimer increased the expression level of.VP1-huFc recombinant plasmids significantly, and the level of exocrine secretion was greatly improved under the guidance of Fc. The expression and secretion of VP1 DNA vaccine with mFc gamma fragment were not ideal. The five New Zealand white rabbits immunized with DNA vaccine found that the immunogenicity of tPA-VP1 was also better, and the level of antibody was highest.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

【参考文献】