本文選題：腸道病毒EV71 + VP1基因��； 參考：《南京醫(yī)科大學(xué)》2011年博士論文

【摘要】：第一部分腸道病毒EV71衣殼蛋白VP1基因的優(yōu)化 目的:為了使腸道病毒EV71的主要中和抗原VP1基因能夠在哺乳動(dòng)物細(xì)胞內(nèi)有效表達(dá),按照哺乳動(dòng)物細(xì)胞密碼子使用的偏好,對(duì)VP1基因序列進(jìn)行密碼子優(yōu)化(codon optimization, opt),并合成優(yōu)化的VP1基因。 方法:選取EV71 BJ4211株VP1基因(Genbank序列號(hào)EU024958.1)序列,用基因分析軟件MacVector 7.2分析其基因編碼序列,找出野生型VP1基因密碼子使用的偏好性,并比較野生型EV71/BJ VP1的基因序列中與哺乳動(dòng)物細(xì)胞密碼子使用偏好不同的位點(diǎn)。對(duì)于與哺乳動(dòng)物細(xì)胞使用偏好相同的密碼子,用哺乳動(dòng)物偏好的密碼子替代野生型EV71/BJ VP1基因中使用偏好不同的密碼子位點(diǎn),然后設(shè)計(jì)出密碼子優(yōu)化的VP1基因。序列的優(yōu)化還包括對(duì)基因mRNA的調(diào)整以使其更穩(wěn)定和更易于在真核細(xì)胞中進(jìn)行轉(zhuǎn)錄和翻譯。密碼子優(yōu)化的VP1基因由德國Geneart公司合成,為便于目的基因的鑒定和插入,在基因兩端分別加上限制性內(nèi)切酶位點(diǎn)PstⅠ和BamHⅠ,裝入骨架質(zhì)粒pGA4,構(gòu)建重組質(zhì)粒pGA4/VP1-opt。將pGA4/VP1-opt轉(zhuǎn)化大腸桿菌HB101,挑取單克隆,小量提取質(zhì)粒鑒定;陽性克隆三次劃氨芐青霉素(Amp)LB平板,獲得含有pGA4/VP1-opt質(zhì)粒的HB101菌種,20%甘油,-70℃保存。 結(jié)果與結(jié)論:經(jīng)酶切證實(shí)插入到載體pGA4中的基因片段大小正確,獲得密碼子優(yōu)化的VP1基因。通過軟件分析發(fā)現(xiàn),與野生型VP1基因相比,密碼子優(yōu)化的VP1基因中哺乳動(dòng)物細(xì)胞偏好的密碼子出現(xiàn)頻率增加,從而使其更適于在哺乳動(dòng)物細(xì)胞中進(jìn)行蛋白表達(dá),但是其編碼VP1蛋白的氨基酸序列不變,以保證原有的抗原構(gòu)象。 目的:為了提高EV71衣殼蛋白VP1基因在真核細(xì)胞的的表達(dá)和分泌水平,增強(qiáng)其免疫原性,以DNA疫苗為工具,通過選擇合適的載體,改造基因表達(dá)和分泌的調(diào)控元件,設(shè)計(jì)并構(gòu)建多種形式的密碼子優(yōu)化的VP1 DNA疫苗。通過分析不同形式的VP1 DNA疫苗在真核細(xì)胞的表達(dá)和分泌水平,及其免疫動(dòng)物刺激產(chǎn)生抗體的能力,比較、評(píng)價(jià)何種形式的VP1蛋白表達(dá)和分泌最佳,具有良好的免疫原性,為篩選有效的EV71VP1蛋白候選疫苗奠定基礎(chǔ)。 方法:將pGA4/VP1-opt質(zhì)粒用PstⅠ和BamHⅠ限制性內(nèi)切酶進(jìn)行雙酶切,通過割膠純化獲得密碼子優(yōu)化的VP1基因,插入到真核表達(dá)載體pJW4303中,構(gòu)建基因優(yōu)化的VP1 DNA疫苗pJW4303/VP1,由于本研究后續(xù)的設(shè)計(jì)均是基于此優(yōu)化的VP1基因,因此這種單純的基因優(yōu)化的VP1 DNA疫苗稱之為“野生型(widetype,即VP1-wt)”。通過設(shè)計(jì)PCR引物P1和P2,以pGA4/VP1-opt質(zhì)粒為模板,擴(kuò)增獲得含有NheⅠ和BamHⅠ酶切位點(diǎn)的VP1基因,克隆到pJW4303載體的tPA信號(hào)肽序列下游,構(gòu)建含有tPA信號(hào)肽的VP1 DNA疫苗pJW4303/tPA-VP1(tPA-VP1),以促進(jìn)蛋白的表達(dá)與分泌。為模擬自然狀態(tài)下VP1易于自聯(lián)形成二聚體的特點(diǎn),設(shè)計(jì)雙聚體VP1 DNA疫苗:合成PCR引物P3和P4,用P1和P4引物PCR擴(kuò)增獲得VP1插入片段1(Insert1,含NheⅠ和KpnⅠ酶切位點(diǎn)),用P3和P2引物PCR擴(kuò)增獲得VP1插入片段2(Insert2,含KpnⅠ和BamHⅠ酶切位點(diǎn)),將Insert1和Insert2同時(shí)克隆到pJW4303載體的tPA信號(hào)肽序列下游,構(gòu)建含有tPA信號(hào)肽的雙聚體VP1 DNA疫苗pJW4303/tPA-VP1-dimer(dimer)。由于免疫球蛋白IgG Fc(Fcγ)片段在機(jī)體免疫中的巨大優(yōu)勢,分別設(shè)計(jì)引物,PCR擴(kuò)增含有KpnⅠ和BamHⅠ酶切位點(diǎn)的人Fcγ(huFcγ)和小鼠Fcγ(mFcγ)基因,將VP1-dimer質(zhì)粒中的Insert2切掉,插入Fcγ基因,構(gòu)建含F(xiàn)cγ基因的VP1 DNA疫苗pJW4303/tPA-VP1-Fcγ(VP1- Fcγ),達(dá)到增強(qiáng)抗原與免疫細(xì)胞的接觸,提高免疫原性的目的。構(gòu)建的重組質(zhì)粒轉(zhuǎn)化大腸桿菌HB101,挑取單克隆,進(jìn)行質(zhì)粒提取、酶切和測序鑒定。鑒定正確的陽性克隆三次劃平板,進(jìn)行甘油菌種保存。 鑒定成功的重組質(zhì)粒進(jìn)行質(zhì)粒大量提取,轉(zhuǎn)染293T細(xì)胞,收獲轉(zhuǎn)染上清和細(xì)胞裂解產(chǎn)物,通過酶聯(lián)免疫標(biāo)記法(ELISA)、間接免疫熒光分析(IFA)和蛋白質(zhì)免疫印跡(Western blot)檢測轉(zhuǎn)染細(xì)胞中VP1蛋白的表達(dá)。提取的質(zhì)粒還用于免疫動(dòng)物,免疫方法采用肌肉注射加電轉(zhuǎn)錄活體基因?qū)敕绞?免疫劑量為200μg/兔。免疫在0、2、4和第8周進(jìn)行,共4次。每次免疫前采血,分離血清,通過檢測血清中特異性IgG抗體的應(yīng)答水平,比較不同形式VP1 DNA疫苗的刺激產(chǎn)生抗體的能力,綜合評(píng)價(jià)疫苗的免疫原性。 結(jié)果和結(jié)論:基于密碼子優(yōu)化的VP1基因,成功設(shè)計(jì)和構(gòu)建了五種形式的VP1 DNA疫苗:VP1-wt、tPA-VP1、VP1-dimer、VP1- huFcγ和VP1- mFcγ。 五種形式的VP1 DNA疫苗均能在真核293T細(xì)胞中表達(dá),加tPA信號(hào)肽后,VP1蛋白表達(dá)和細(xì)胞外分泌水平均明顯增加;VP1-dimer的表達(dá)和分泌水平則進(jìn)一步提高。VP1-huFc重組質(zhì)粒不僅表達(dá)水平顯著增加,其在Fc的引導(dǎo)下,細(xì)胞外分泌水平也大大提高。而加有mFcγ片段的VP1 DNA疫苗的表達(dá)和分泌則并不理想。五種DNA疫苗免疫新西蘭白兔發(fā)現(xiàn),tPA-VP1免疫原性也較好,產(chǎn)生抗體水平最高。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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