內含HIV-1 gag編碼mRNA的MS2病毒樣顆粒構建及體外表達和體內免疫應答研究
發(fā)布時間：2018-08-05 09:53
【摘要】：研究背景 RNA不會整合到宿主細胞基因組,也不會引起自身免疫病或者產(chǎn)生抗DNA抗體等嚴重的副作用。從安全性角度考慮,基于mRNA的基因疫苗要優(yōu)于基于DNA的疫苗。但是mRNA容易降解、穩(wěn)定性差,且體內轉移效率低,這無疑限制了mRNA的使用。MS2病毒樣顆粒穩(wěn)定性高,能夠保護裝載的外源mRNA,展示了其作為RNA疫苗新型遞送載體的潛在應用價值。然而,真核系統(tǒng)表達的MS2病毒樣顆粒(VLPs)能否在動物水平有效地誘導抗原特異性免疫應答,在國內和國際上還沒有報道。 研究目的 本研究通過釀酒酵母細胞表達系統(tǒng)獲得裝載HIV-1 gag編碼mRNA的MS2VLPs。從體外細胞水平驗證MS2 VLPs包裝的mRNA能否在哺乳動物細胞內發(fā)揮功能,并通過體內免疫動物驗證MS2 VLPs能否誘導抗原特異性的免疫應答。 方法 1.單質粒雙表達重組載體的構建：將編碼衣殼蛋白的cDNA序列插入pESC-Ura載體,構建衣殼蛋白載體pMS。將編碼HIV-1 GAG蛋白的cDNA序列插入pMS構建能包裝HIV-1 Gag mRNA的pMS-GAG VLPs的表達載體。構建能包裝MS2衣殼蛋白自身編碼：mRNA的pMS2C VLPs的表達載體作為對照。 2. VLPs的表達：上述構建好的pMS-GAG載體和pMS2C載體經(jīng)測序鑒定正確后,轉化到釀酒酵母YPH499細胞內。挑取陽性單克隆細胞在SD培養(yǎng)基中擴大培養(yǎng),然后SG培養(yǎng)基誘導表達MS2病毒樣顆粒。 3. VLPs的純化：超聲波碎酵母后獲得含有HIV-1 gag mRNA的重組MS2病毒樣顆粒。在上清中加入DNase I和RNase A,37℃過夜消化,然后分子篩色譜層析純化VLPs。 4. VLPs的驗證：采用瓊脂糖凝膠電泳、十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)及電子顯微鏡鑒定。對上述純化后的VLPs進行RT-PCR擴增,以驗證VLPs是否包裝了全長的HIV-1 gag RNA。 5. pMS-GAG VLPs穩(wěn)定性實驗和耐酶性實驗。 6. VLPs包裝HIV-1 gag RNA在哺乳動物細胞系的表達：提取pMS-GAG VLPs包裝的RNA,使用DOTAP轉染試劑轉染至哺乳動物細胞。24 h后,收集細胞,破碎后使用Genescreen Plus HIV Ag-Ab Elisa檢測試劑盒檢測gag蛋白的表達。 7. MS2 VLPs免疫小鼠：取15μg純化的pMS-GAG VLPs (5×1012 RNA拷貝)混合完全弗氏佐劑(佐劑和抗原體積比為1：1)制備成”油包水”乳狀液,免疫6-8周BALB/c小鼠。第3周和第5周使用同樣的VLPs混合不完全弗氏佐劑,加強免疫兩次。以15μg純化的pMS2C VLPs作為對照。最后一次免疫后1周,取小鼠尾靜脈血檢測抗原特異性抗體的產(chǎn)生。 結果 利用單質粒表達系統(tǒng),在釀酒酵母YPH499細胞內MS2衣殼蛋白成功的包裝了HIV-1 gag mRNA,形成了MS2 VLPs。DNase和RNase消化結果表明,此病毒樣顆粒具有很好的耐DNase和RNase的特性。提取RNA轉染哺乳動物細胞(293T細胞、CHO細胞)后可檢測到目的蛋白的表達。我們還進一步證實了純化后的pMS-GAG VLPs免疫BALB/c (H-2d)小鼠后可以誘導抗原特異性體液免疫應答。 結論 1.應用傳統(tǒng)分子克隆程序,成功構建MS2病毒樣顆粒的釀酒酵母表達載體pMS-GAG。經(jīng)瓊脂糖凝膠電泳、十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(SDS-PAGE)及電子顯微鏡等檢測的方法證實釀酒酵母細胞表達系統(tǒng)可成功地表達MS2 VLPs。 2.通過釀酒酵母表達系統(tǒng)獲得的MS2病毒樣顆粒能夠保護包裝的RNA不被降解,而且穩(wěn)定性好,可在40℃儲存四個月以上。MS2病毒樣顆粒具有無潛在生物傳染危險性、易生產(chǎn)、運輸和使用方便的優(yōu)點。這些特性使得MS2 VLPs滿足RNA遞送載體的要求。 3.通過釀酒酵母表達系統(tǒng)獲得的MS2病毒樣顆粒包裝的mRNA在真核系統(tǒng)中具有功能性并能夠被翻譯成蛋白。本課題在國內外首次證實釀酒酵母細胞表達的MS2 VLPs可作為mRNA的新型遞送載體,內含HIV-1 gag編碼mRNA的MS2病毒樣顆粒免疫小鼠后可誘導針對HIV-1 gag蛋白的特異性抗體的產(chǎn)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
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392.1
本文編號：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
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392.1
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