内含HIV-1 gag编码mRNA的MS2病毒样颗粒构建及体外表达和体内免疫应答研究
发布时间：2018-08-05 09:53
【摘要】：研究背景 RNA不会整合到宿主细胞基因组,也不会引起自身免疫病或者产生抗DNA抗体等严重的副作用。从安全性角度考虑,基于mRNA的基因疫苗要优于基于DNA的疫苗。但是mRNA容易降解、稳定性差,且体内转移效率低,这无疑限制了mRNA的使用。MS2病毒样颗粒稳定性高,能够保护装载的外源mRNA,展示了其作为RNA疫苗新型递送载体的潜在应用价值。然而,真核系统表达的MS2病毒样颗粒(VLPs)能否在动物水平有效地诱导抗原特异性免疫应答,在国内和国际上还没有报道。 研究目的 本研究通过酿酒酵母细胞表达系统获得装载HIV-1 gag编码mRNA的MS2VLPs。从体外细胞水平验证MS2 VLPs包装的mRNA能否在哺乳动物细胞内发挥功能,并通过体内免疫动物验证MS2 VLPs能否诱导抗原特异性的免疫应答。 方法 1.单质粒双表达重组载体的构建：将编码衣壳蛋白的cDNA序列插入pESC-Ura载体,构建衣壳蛋白载体pMS。将编码HIV-1 GAG蛋白的cDNA序列插入pMS构建能包装HIV-1 Gag mRNA的pMS-GAG VLPs的表达载体。构建能包装MS2衣壳蛋白自身编码：mRNA的pMS2C VLPs的表达载体作为对照。 2. VLPs的表达：上述构建好的pMS-GAG载体和pMS2C载体经测序鉴定正确后,转化到酿酒酵母YPH499细胞内。挑取阳性单克隆细胞在SD培养基中扩大培养,然后SG培养基诱导表达MS2病毒样颗粒。 3. VLPs的纯化：超声波碎酵母后获得含有HIV-1 gag mRNA的重组MS2病毒样颗粒。在上清中加入DNase I和RNase A,37℃过夜消化,然后分子筛色谱层析纯化VLPs。 4. VLPs的验证：采用琼脂糖凝胶电泳、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及电子显微镜鉴定。对上述纯化后的VLPs进行RT-PCR扩增,以验证VLPs是否包装了全长的HIV-1 gag RNA。 5. pMS-GAG VLPs稳定性实验和耐酶性实验。 6. VLPs包装HIV-1 gag RNA在哺乳动物细胞系的表达：提取pMS-GAG VLPs包装的RNA,使用DOTAP转染试剂转染至哺乳动物细胞。24 h后,收集细胞,破碎后使用Genescreen Plus HIV Ag-Ab Elisa检测试剂盒检测gag蛋白的表达。 7. MS2 VLPs免疫小鼠：取15μg纯化的pMS-GAG VLPs (5×1012 RNA拷贝)混合完全弗氏佐剂(佐剂和抗原体积比为1：1)制备成”油包水”乳状液,免疫6-8周BALB/c小鼠。第3周和第5周使用同样的VLPs混合不完全弗氏佐剂,加强免疫两次。以15μg纯化的pMS2C VLPs作为对照。最后一次免疫后1周,取小鼠尾静脉血检测抗原特异性抗体的产生。 结果 利用单质粒表达系统,在酿酒酵母YPH499细胞内MS2衣壳蛋白成功的包装了HIV-1 gag mRNA,形成了MS2 VLPs。DNase和RNase消化结果表明,此病毒样颗粒具有很好的耐DNase和RNase的特性。提取RNA转染哺乳动物细胞(293T细胞、CHO细胞)后可检测到目的蛋白的表达。我们还进一步证实了纯化后的pMS-GAG VLPs免疫BALB/c (H-2d)小鼠后可以诱导抗原特异性体液免疫应答。 结论 1.应用传统分子克隆程序,成功构建MS2病毒样颗粒的酿酒酵母表达载体pMS-GAG。经琼脂糖凝胶电泳、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及电子显微镜等检测的方法证实酿酒酵母细胞表达系统可成功地表达MS2 VLPs。 2.通过酿酒酵母表达系统获得的MS2病毒样颗粒能够保护包装的RNA不被降解,而且稳定性好,可在40℃储存四个月以上。MS2病毒样颗粒具有无潜在生物传染危险性、易生产、运输和使用方便的优点。这些特性使得MS2 VLPs满足RNA递送载体的要求。 3.通过酿酒酵母表达系统获得的MS2病毒样颗粒包装的mRNA在真核系统中具有功能性并能够被翻译成蛋白。本课题在国内外首次证实酿酒酵母细胞表达的MS2 VLPs可作为mRNA的新型递送载体,内含HIV-1 gag编码mRNA的MS2病毒样颗粒免疫小鼠后可诱导针对HIV-1 gag蛋白的特异性抗体的产生
[Abstract]:Research background
RNA does not integrate into the host cell genome, nor does it cause serious side effects such as autoimmune disease or anti DNA antibody. From the security point of view, the mRNA based gene vaccine is superior to the DNA based vaccine. But mRNA is easily degraded, the stability is poor, and the transfer efficiency in the body is low, which undoubtedly restricts the use of the.MS2 virus like mRNA. It has high stability and can protect the loaded exogenous mRNA, which shows its potential application value as a new delivery carrier for RNA vaccine. However, whether the MS2 virus like particles (VLPs) expressed in the eukaryotic system can effectively induce antigen specific immune response at animal level has not been reported at home and abroad.
research objective
In this study, the MS2VLPs. of HIV-1 gag encoded mRNA was obtained from the yeast cell expression system to verify whether the mRNA in the MS2 VLPs package can function in the mammalian cells, and the immune animals of the body verify whether MS2 VLPs can induce the antigen specific immune response.
Method
1. the construction of the recombinant vector of the single grain double expression: inserting the cDNA sequence of the capsid protein into the pESC-Ura vector, constructing the capsid protein carrier pMS. and inserting the cDNA sequence encoding the HIV-1 GAG protein into pMS to construct the pMS-GAG VLPs, which can package the HIV-1 Gag mRNA. The expression vector was used as a control.
2. VLPs expression: the constructed pMS-GAG vector and pMS2C vector were correctly sequenced and transformed into Saccharomyces cerevisiae YPH499 cells. The positive monoclonal cells were picked up in the SD medium to expand culture, and then the SG medium induced the expression of MS2 virus like particles.
3. VLPs purification: after ultrasonic crushing yeast, the recombinant MS2 virus particles containing HIV-1 gag mRNA were obtained. DNase I and RNase A were added to the supernatant, at 37 C for overnight digestion, and then VLPs. was purified by molecular sieve chromatography.
4. VLPs verification: agarose gel electrophoresis, twelve alkyl sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electronic microscope identification. The purified VLPs was amplified by RT-PCR to verify that VLPs packaged the full length of HIV-1 gag RNA..
5. pMS-GAG VLPs stability experiment and enzyme resistance test.
6. VLPs packaging HIV-1 gag RNA was expressed in mammalian cell lines: RNA was extracted from pMS-GAG VLPs, and DOTAP transfected reagents were transfected to.24 h of mammalian cells, and cells were collected.
7. MS2 VLPs immunized mice: 15 u g purified pMS-GAG VLPs (5 x 1012 RNA copies) mixed complete Freund's adjuvant (adjuvant and antigen volume ratio) were prepared for "oil wrapped water" emulsion, immunized for 6-8 weeks BALB/c mice. The same VLPs mixed incomplete Freund adjuvant was used for third and fifth weeks, and immune two was strengthened. PMS2C V was purified with 15 micron G. LPs was used as a control. 1 weeks after the last immunization, the tail vein blood of mice was taken to detect the production of antigen specific antibodies.
Result
The MS2 capsid protein in Saccharomyces cerevisiae YPH499 cells successfully packaged HIV-1 gag mRNA in Saccharomyces cerevisiae cells by using the mono granular expression system. The results of MS2 VLPs.DNase and RNase digestion showed that the virus like particles had good properties of DNase and RNase. The target protein could be detected after the RNA transfection of mammalian cells (293T cells). We further confirmed that the purified pMS-GAG VLPs could induce antigen-specific humoral immune responses in BALB/c (H-2d) mice.
conclusion
1. using the traditional molecular cloning program, the yeast expression vector pMS-GAG. with MS2 virus like particles was successfully constructed by agarose gel electrophoresis, twelve alkyl sodium polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy, which proved that the Saccharomyces cerevisiae cell expression system can express MS2 VLPs. successfully.
2. the MS2 virus like particles obtained through the Saccharomyces cerevisiae expression system can protect the packaged RNA from degradation, and have good stability, and can be stored at 40 centigrade for more than four months. The.MS2 virus like particles have the advantages of no potential biotic risk, easy production, transportation and use, which make MS2 VLPs meet the requirement of RNA delivery carrier. Ask.
3. the mRNA of MS2 virus like particles obtained by Saccharomyces cerevisiae expressed in the eukaryotic system is functional and can be translated into protein. This topic is first confirmed at home and abroad for the first time that the MS2 VLPs expressed by Saccharomyces cerevisiae can be used as a new delivery carrier of mRNA, and the MS2 virus like particles containing HIV-1 gag encoded mRNA are immune to mice. Induction of specific antibodies against HIV-1 Gag protein
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2165424
[Abstract]:Research background
RNA does not integrate into the host cell genome, nor does it cause serious side effects such as autoimmune disease or anti DNA antibody. From the security point of view, the mRNA based gene vaccine is superior to the DNA based vaccine. But mRNA is easily degraded, the stability is poor, and the transfer efficiency in the body is low, which undoubtedly restricts the use of the.MS2 virus like mRNA. It has high stability and can protect the loaded exogenous mRNA, which shows its potential application value as a new delivery carrier for RNA vaccine. However, whether the MS2 virus like particles (VLPs) expressed in the eukaryotic system can effectively induce antigen specific immune response at animal level has not been reported at home and abroad.
research objective
In this study, the MS2VLPs. of HIV-1 gag encoded mRNA was obtained from the yeast cell expression system to verify whether the mRNA in the MS2 VLPs package can function in the mammalian cells, and the immune animals of the body verify whether MS2 VLPs can induce the antigen specific immune response.
Method
1. the construction of the recombinant vector of the single grain double expression: inserting the cDNA sequence of the capsid protein into the pESC-Ura vector, constructing the capsid protein carrier pMS. and inserting the cDNA sequence encoding the HIV-1 GAG protein into pMS to construct the pMS-GAG VLPs, which can package the HIV-1 Gag mRNA. The expression vector was used as a control.
2. VLPs expression: the constructed pMS-GAG vector and pMS2C vector were correctly sequenced and transformed into Saccharomyces cerevisiae YPH499 cells. The positive monoclonal cells were picked up in the SD medium to expand culture, and then the SG medium induced the expression of MS2 virus like particles.
3. VLPs purification: after ultrasonic crushing yeast, the recombinant MS2 virus particles containing HIV-1 gag mRNA were obtained. DNase I and RNase A were added to the supernatant, at 37 C for overnight digestion, and then VLPs. was purified by molecular sieve chromatography.
4. VLPs verification: agarose gel electrophoresis, twelve alkyl sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electronic microscope identification. The purified VLPs was amplified by RT-PCR to verify that VLPs packaged the full length of HIV-1 gag RNA..
5. pMS-GAG VLPs stability experiment and enzyme resistance test.
6. VLPs packaging HIV-1 gag RNA was expressed in mammalian cell lines: RNA was extracted from pMS-GAG VLPs, and DOTAP transfected reagents were transfected to.24 h of mammalian cells, and cells were collected.
7. MS2 VLPs immunized mice: 15 u g purified pMS-GAG VLPs (5 x 1012 RNA copies) mixed complete Freund's adjuvant (adjuvant and antigen volume ratio) were prepared for "oil wrapped water" emulsion, immunized for 6-8 weeks BALB/c mice. The same VLPs mixed incomplete Freund adjuvant was used for third and fifth weeks, and immune two was strengthened. PMS2C V was purified with 15 micron G. LPs was used as a control. 1 weeks after the last immunization, the tail vein blood of mice was taken to detect the production of antigen specific antibodies.
Result
The MS2 capsid protein in Saccharomyces cerevisiae YPH499 cells successfully packaged HIV-1 gag mRNA in Saccharomyces cerevisiae cells by using the mono granular expression system. The results of MS2 VLPs.DNase and RNase digestion showed that the virus like particles had good properties of DNase and RNase. The target protein could be detected after the RNA transfection of mammalian cells (293T cells). We further confirmed that the purified pMS-GAG VLPs could induce antigen-specific humoral immune responses in BALB/c (H-2d) mice.
conclusion
1. using the traditional molecular cloning program, the yeast expression vector pMS-GAG. with MS2 virus like particles was successfully constructed by agarose gel electrophoresis, twelve alkyl sodium polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy, which proved that the Saccharomyces cerevisiae cell expression system can express MS2 VLPs. successfully.
2. the MS2 virus like particles obtained through the Saccharomyces cerevisiae expression system can protect the packaged RNA from degradation, and have good stability, and can be stored at 40 centigrade for more than four months. The.MS2 virus like particles have the advantages of no potential biotic risk, easy production, transportation and use, which make MS2 VLPs meet the requirement of RNA delivery carrier. Ask.
3. the mRNA of MS2 virus like particles obtained by Saccharomyces cerevisiae expressed in the eukaryotic system is functional and can be translated into protein. This topic is first confirmed at home and abroad for the first time that the MS2 VLPs expressed by Saccharomyces cerevisiae can be used as a new delivery carrier of mRNA, and the MS2 virus like particles containing HIV-1 gag encoded mRNA are immune to mice. Induction of specific antibodies against HIV-1 Gag protein
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1
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