豬腦心肌炎病毒NJ08株感染性cDNA克隆構(gòu)建及拯救病毒生物學(xué)特性
發(fā)布時(shí)間：2018-12-15 11:57
【摘要】：腦心肌炎病毒(Encephalomyocarditis virus,EMCV)屬于微RNA病毒科心病毒屬成員,是一種無囊膜、單股正鏈的RNA病毒,是一種重要的人畜共患病病原體。EMCV感染后可造成仔豬急性致死性心肌炎,致死率可高達(dá)100%。本研究以我國中東地區(qū)EMCV NJ08分離株為材料,采用體內(nèi)轉(zhuǎn)錄的方法成功建立了此毒株的反向遺傳操作系統(tǒng),成功拯救到病毒,并對(duì)該病毒的生物學(xué)特性進(jìn)行鑒定;拯救病毒的免疫原性也得到初步的研究,為今后深入研究腦心肌炎病毒的分子致病機(jī)制、基因功能以及新型疫苗奠定了基礎(chǔ)。具體研究內(nèi)容如下:1豬腦心肌炎病毒感染性克隆的構(gòu)建及鑒定本研究采用RT-PCR方法獲得覆蓋EMCV NJ08株全基因組(GenBank HM641897)的A、B和C三個(gè)基因片段,其中C片段第6243位的T突變?yōu)锳,引入EcoRI標(biāo)記位點(diǎn),分別克隆至pEasy simple Blunt載體,基因測序正確。采用PacI、XhoI、NheI和SpeI將3個(gè)片段依次克隆至CMY-β質(zhì)粒,獲得含EMCV全基因組cDNA的重組質(zhì)粒pCMV-NJ08。采用脂質(zhì)體將其轉(zhuǎn)染至BHK-21細(xì)胞,結(jié)果出現(xiàn)明顯細(xì)胞病變,經(jīng)RT-PCR產(chǎn)物EcoRI酶切和間接免疫熒光試驗(yàn)鑒定,證明獲得重組EMCV rNJ08;拯救病毒rNJ08株在BHK21細(xì)胞上的TCID_(50)、一步生長曲線和空斑形態(tài)與親本毒NJ08相似,但對(duì)小鼠致病性明顯降低,為該病毒致病機(jī)制及疫苗研制奠定了重要基礎(chǔ)。2重組腦心肌炎病毒滅活疫苗小鼠免疫特性本研究使用終濃度為0.002mM的BEI將重組病毒(rNJ08)和親本病毒(NJ08)進(jìn)行滅活處理,加入ISA15A和ISA206佐劑分別配制成滅活疫苗。將6周齡ICR小鼠隨機(jī)分成8組,每組5只。第1、2組分別皮下注射由ISA15A佐劑配制的NJ08和rNJ08滅活疫苗,第3、4組分別皮下注射由ISA206佐劑配制的NJ08和rNJ08滅活疫苗(抗原濃度均為10~7 TCID_(50)/0.2ml)。第5-7組依次皮下多點(diǎn)注射等體積的由ISA15A佐劑配制的梯度抗原濃度(10~6TCID_(50)/0.2ml、10~5 TCID_(50)/0.2ml、10~4TCID_(50)/0.2ml)的 rNJ08滅活疫苗;第8組皮下注射2%DMEM作為陰性對(duì)照。間隔三周進(jìn)行加強(qiáng)免疫。首免后3周采集小鼠血清測其ELISA抗體水平;二免后3周采集血清測定ELISA抗體效價(jià)和中和抗體效價(jià),同時(shí)分離脾臟淋巴細(xì)胞進(jìn)行淋巴細(xì)胞增殖轉(zhuǎn)化試驗(yàn)。結(jié)果為:rNJ08疫苗免疫組ELISA和中和抗體水平具有明顯的抗原劑量依賴性關(guān)系;各免疫組小鼠血清ELISA和中和抗體水平在加強(qiáng)免疫后顯著提高(1:16以上)·淋巴細(xì)胞增殖試驗(yàn)結(jié)果顯示,接種相同劑量的各免疫組刺激指數(shù)均在3.0左右。結(jié)果表明rNJ08與NJ08具有相似的免疫原性,均能有效地刺激機(jī)體產(chǎn)生良好的免疫反應(yīng),并能產(chǎn)生中和抗體。由于該重組病毒毒力較弱,因此,安全性更高。
[Abstract]:Encephalitis virus (Encephalomyocarditis virus,EMCV), a member of the family RNA virus family, is a non-encapsulated, single-stranded RNA virus, which is an important zoonotic pathogen. EMCV infection can cause acute fatal myocarditis in piglets. Mortality can be as high as 100. In this study, the reverse genetic operating system of EMCV NJ08 isolate from the Middle East region of China was successfully established by in vivo transcription method, and the virus was successfully saved, and the biological characteristics of the virus were identified. The immunogenicity of the rescue virus has also been preliminarily studied, which will lay a foundation for further study on the molecular pathogenesis, gene function and new vaccine of encephalitis virus. The specific contents of the study were as follows: 1 Construction and identification of infectious clone of porcine encephalomyocarditis virus. In this study, three gene fragments, Agna B and C, covering the whole genome (GenBank HM641897) of EMCV NJ08 strain were obtained by RT-PCR method. The T mutation at position 6243 of the C fragment was A. the EcoRI marker site was introduced and cloned into the pEasy simple Blunt vector, and the gene was sequenced correctly. The three fragments were cloned into CMY- 尾 plasmid by PacI,XhoI,NheI and SpeI, and the recombinant plasmid pCMV-NJ08. containing EMCV genomic cDNA was obtained. After transfection with liposome into BHK-21 cells, obvious cytopathic changes were observed. The recombinant EMCV rNJ08; was confirmed by EcoRI digestion and indirect immunofluorescence assay. The one-step growth curve and plaque morphology of TCID_ (50) on BHK21 cells were similar to those of parent virus NJ08, but the pathogenicity of TCID_ (50) on BHK21 cells was significantly decreased. 2 the immune characteristics of recombinant encephalitis virus inactivated vaccine in mice. In this study, the recombinant virus (rNJ08) and parental virus (NJ08) were inactivated by BEI with the final concentration of 0.002mM. Inactivated vaccine was prepared by adding ISA15A and ISA206 adjuvant respectively. ICR mice aged 6 weeks were randomly divided into 8 groups with 5 mice in each group. NJ08 and rNJ08 inactivated vaccine prepared by ISA15A adjuvant were injected subcutaneously into the first group and NJ08 and rNJ08 inactivated vaccine prepared by ISA206 adjuvant were injected subcutaneously in the third group (antigen concentration was 10 7 TCID_ (50) / 0.2ml). In group 5-7, the gradient antigen concentration (10 ~ 6TCID _ (50) / 0.2ml of CID _ (50) / 0.2ml ~ 104TCID _ (50) / 0.2ml) was injected subcutaneously with the same volume of rNJ08 inactivated vaccine, and the dose of gradient antigen (10 ~ 6TCID _ (50) / 0.2ml) was 104TCID _ (50) / 0.2ml. Group 8 was subcutaneously injected with 2%DMEM as negative control. The immunization was strengthened at intervals of three weeks. The levels of ELISA antibody in serum of mice were measured 3 weeks after the first immunization, and the titers of ELISA antibody and neutralizing antibody were measured 3 weeks after the second immunization, and lymphocyte proliferation and transformation tests were performed by isolating spleen lymphocytes at the same time. The results showed that the levels of ELISA and neutralizing antibody in rNJ08 vaccine immunized group were in a dose-dependent manner. The levels of serum ELISA and neutralizing antibody in each immunized group were significantly increased (more than 1:16) after enhanced immunization. The results of lymphocyte proliferation test showed that the stimulating index of the same dose of each immunization group was about 3. 0. The results showed that rNJ08 and NJ08 had similar immunogenicity, which could effectively stimulate the body to produce a good immune response and produce neutralizing antibodies. Because of the weak virulence of the recombinant virus, the safety of the recombinant virus is higher.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
本文編號(hào)：2380593
[Abstract]:Encephalitis virus (Encephalomyocarditis virus,EMCV), a member of the family RNA virus family, is a non-encapsulated, single-stranded RNA virus, which is an important zoonotic pathogen. EMCV infection can cause acute fatal myocarditis in piglets. Mortality can be as high as 100. In this study, the reverse genetic operating system of EMCV NJ08 isolate from the Middle East region of China was successfully established by in vivo transcription method, and the virus was successfully saved, and the biological characteristics of the virus were identified. The immunogenicity of the rescue virus has also been preliminarily studied, which will lay a foundation for further study on the molecular pathogenesis, gene function and new vaccine of encephalitis virus. The specific contents of the study were as follows: 1 Construction and identification of infectious clone of porcine encephalomyocarditis virus. In this study, three gene fragments, Agna B and C, covering the whole genome (GenBank HM641897) of EMCV NJ08 strain were obtained by RT-PCR method. The T mutation at position 6243 of the C fragment was A. the EcoRI marker site was introduced and cloned into the pEasy simple Blunt vector, and the gene was sequenced correctly. The three fragments were cloned into CMY- 尾 plasmid by PacI,XhoI,NheI and SpeI, and the recombinant plasmid pCMV-NJ08. containing EMCV genomic cDNA was obtained. After transfection with liposome into BHK-21 cells, obvious cytopathic changes were observed. The recombinant EMCV rNJ08; was confirmed by EcoRI digestion and indirect immunofluorescence assay. The one-step growth curve and plaque morphology of TCID_ (50) on BHK21 cells were similar to those of parent virus NJ08, but the pathogenicity of TCID_ (50) on BHK21 cells was significantly decreased. 2 the immune characteristics of recombinant encephalitis virus inactivated vaccine in mice. In this study, the recombinant virus (rNJ08) and parental virus (NJ08) were inactivated by BEI with the final concentration of 0.002mM. Inactivated vaccine was prepared by adding ISA15A and ISA206 adjuvant respectively. ICR mice aged 6 weeks were randomly divided into 8 groups with 5 mice in each group. NJ08 and rNJ08 inactivated vaccine prepared by ISA15A adjuvant were injected subcutaneously into the first group and NJ08 and rNJ08 inactivated vaccine prepared by ISA206 adjuvant were injected subcutaneously in the third group (antigen concentration was 10 7 TCID_ (50) / 0.2ml). In group 5-7, the gradient antigen concentration (10 ~ 6TCID _ (50) / 0.2ml of CID _ (50) / 0.2ml ~ 104TCID _ (50) / 0.2ml) was injected subcutaneously with the same volume of rNJ08 inactivated vaccine, and the dose of gradient antigen (10 ~ 6TCID _ (50) / 0.2ml) was 104TCID _ (50) / 0.2ml. Group 8 was subcutaneously injected with 2%DMEM as negative control. The immunization was strengthened at intervals of three weeks. The levels of ELISA antibody in serum of mice were measured 3 weeks after the first immunization, and the titers of ELISA antibody and neutralizing antibody were measured 3 weeks after the second immunization, and lymphocyte proliferation and transformation tests were performed by isolating spleen lymphocytes at the same time. The results showed that the levels of ELISA and neutralizing antibody in rNJ08 vaccine immunized group were in a dose-dependent manner. The levels of serum ELISA and neutralizing antibody in each immunized group were significantly increased (more than 1:16) after enhanced immunization. The results of lymphocyte proliferation test showed that the stimulating index of the same dose of each immunization group was about 3. 0. The results showed that rNJ08 and NJ08 had similar immunogenicity, which could effectively stimulate the body to produce a good immune response and produce neutralizing antibodies. Because of the weak virulence of the recombinant virus, the safety of the recombinant virus is higher.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65
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