猴免疫缺陷病毒p27基因的克隆、表达、单克隆抗体制备及鉴定
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摘要
研究目的：人免疫缺陷病毒(Human immunodeficiency virus, HIV)与猴免疫缺陷病毒(Simian immunodeficiency virus, SIV)具有亲缘关系，均属于逆转录病毒科慢病毒属。SIV/恒河猴动物模型被广泛应用于AIDS的研究中。HIV p24蛋白是核衣壳蛋白，也是病毒的主要抗原成分,常用于病毒的定性定量检测。对应在SIV的p27蛋白，也常用于动物或体外细胞中检测SIV的感染。本文旨在利用基因工程的方法获得高纯度的p27抗原，制备抗p27单克隆抗体，用于检测细胞和组织中SIV感染情况。
研究方法：从SIVmac251基因组提取RNA，以随机引物进行逆转录反应，合成cDNA。PCR按照常规方法进行。根据GenBank中猴免疫缺陷病毒SIV全基因组序列的p27蛋白的基因序列(M19499.1)合成引物：正向引物5’-GGA TCC ATG CCG AGA ACA TTA AAT GGG-3’，反向引物5’-GTC GAC TGC CAT TAA TCT AGCCTT CTG-3’。回收PCR产物，克隆至pMD18-T载体，筛选阳性克隆送测序。提取pMD18-p27和pET-23a质粒分别用BamHⅠ和SalⅠ双酶切，琼脂糖凝胶电泳回收目的片段，将目的基因和载体连接，构建pET-p27质粒，转化大肠杆菌DH5α，菌落PCR鉴定阳性克隆,抽提质粒DNA,用BamH I和Sal I双酶切进一步鉴定并转化大肠杆菌BL21(DE3)plysS。接种含重组表达质粒的大肠杆菌BL21(DE3)plysS于培养基中培养诱导,分析目的蛋白的表达状况。利用亲和层析技术纯化目的蛋白，免疫印迹检测其活性。用纯化后的p27蛋白免疫小鼠。获取其脾细胞，在PEG作用下与骨髓瘤细胞SP2/0融合。经过选择性培养后，筛选产生p27单抗的杂交瘤细胞株，间接ELISA法测定McAb的效价。利用免疫组化技术、流式细胞术分别检测经SIVmac251感染后CEM×174细胞内的病毒抗原。
研究结果：成功表达并纯化了p27蛋白。获得6株稳定分泌抗SIV p27单克隆抗体杂交瘤细胞株。能识别感染SIV的猴子血清中的病毒。其中2株能用于免疫组化实验，1株用于细胞内染色流式分析。
研究结论：成功克隆猴免疫缺陷病毒p27基因，构建原核表达载体pET-p27，在大肠杆菌BL21(DE3)plysS中高效表达出p27蛋白。成功制备了抗SIV p27蛋白的单克隆抗体，为后期检测感染SIV的动物实验奠定基础。
Objective: Human immunodeficiency virus (HIV) and simian immunodeficiencyvirus (SIV) are lentivirus and have an evolutionary relationship. Rhesusmonkey infected with SIV is a widely used animal model in AIDS studies.The p24protein of HIV caspid is a main antigenic component of the virusand commonly used in the qualitative and quantitative detection of HIV.The p27protein of SIV is also commonly used to detect SIV infection inanimals or in vitro studies. This study was designed to use geneticengineering methods to obtain high purity of p27antigen and to prepareanti-p27monoclonal antibody and also to detect SIV antigen in cells andanimal tissues.
Methods: SIVmac251genome RNA was extracted and cDNA was synthesized byreverse transcription reactions with random primers. According to p27gene sequences (GenBank M19499.1), PCR primers were designed asfollowed: the forward primer5'-GGA TCC ATG CCG AGA ACA TTAAAT GGG-3'and the reverse primer5'-GTC GAC TGC CAT TAA TCTAGC CTT CTG-3'. The PCR product was cloned into pMD18-T vectorand sequenced. Positive product was subcloned to pET-23a plasmid andtransformed into E. coli BL21(DE3)plysS. Target protein expression wasinduced by IPTG and purified by His resin affinity chromatography, andthen was identified by Western blotting. Then Blab/c mice wereimmunized with the purified p27protein for three times. The spleen cellswere fused with myeloma cell line SP2/0. Indirect ELISA was used to detect the titers of p27McAbs produced by hybridoma cells.Immunohistochemistry and flow cytometry were used to detect viralantigens in CEM×174cells infected with SIVmac251.
Results: p27protein was successfully expressed and purified and six hybridomacell lines stably-secreting anti-p27monoclonal antibody were obtained.Two of6kinds antibodies could be used for immunohistochemistryexperiments, and another one for flow cytometric analysis of intracellularstaining.
Conclusions: Anti-SIV p27protein monoclonal antibody was successfully prepared,which layed the foundation for the future detection of SIV infection inanimal experiments.
研究方法：从SIVmac251基因组提取RNA，以随机引物进行逆转录反应，合成cDNA。PCR按照常规方法进行。根据GenBank中猴免疫缺陷病毒SIV全基因组序列的p27蛋白的基因序列(M19499.1)合成引物：正向引物5’-GGA TCC ATG CCG AGA ACA TTA AAT GGG-3’，反向引物5’-GTC GAC TGC CAT TAA TCT AGCCTT CTG-3’。回收PCR产物，克隆至pMD18-T载体，筛选阳性克隆送测序。提取pMD18-p27和pET-23a质粒分别用BamHⅠ和SalⅠ双酶切，琼脂糖凝胶电泳回收目的片段，将目的基因和载体连接，构建pET-p27质粒，转化大肠杆菌DH5α，菌落PCR鉴定阳性克隆,抽提质粒DNA,用BamH I和Sal I双酶切进一步鉴定并转化大肠杆菌BL21(DE3)plysS。接种含重组表达质粒的大肠杆菌BL21(DE3)plysS于培养基中培养诱导,分析目的蛋白的表达状况。利用亲和层析技术纯化目的蛋白，免疫印迹检测其活性。用纯化后的p27蛋白免疫小鼠。获取其脾细胞，在PEG作用下与骨髓瘤细胞SP2/0融合。经过选择性培养后，筛选产生p27单抗的杂交瘤细胞株，间接ELISA法测定McAb的效价。利用免疫组化技术、流式细胞术分别检测经SIVmac251感染后CEM×174细胞内的病毒抗原。
研究结果：成功表达并纯化了p27蛋白。获得6株稳定分泌抗SIV p27单克隆抗体杂交瘤细胞株。能识别感染SIV的猴子血清中的病毒。其中2株能用于免疫组化实验，1株用于细胞内染色流式分析。
研究结论：成功克隆猴免疫缺陷病毒p27基因，构建原核表达载体pET-p27，在大肠杆菌BL21(DE3)plysS中高效表达出p27蛋白。成功制备了抗SIV p27蛋白的单克隆抗体，为后期检测感染SIV的动物实验奠定基础。
Objective: Human immunodeficiency virus (HIV) and simian immunodeficiencyvirus (SIV) are lentivirus and have an evolutionary relationship. Rhesusmonkey infected with SIV is a widely used animal model in AIDS studies.The p24protein of HIV caspid is a main antigenic component of the virusand commonly used in the qualitative and quantitative detection of HIV.The p27protein of SIV is also commonly used to detect SIV infection inanimals or in vitro studies. This study was designed to use geneticengineering methods to obtain high purity of p27antigen and to prepareanti-p27monoclonal antibody and also to detect SIV antigen in cells andanimal tissues.
Methods: SIVmac251genome RNA was extracted and cDNA was synthesized byreverse transcription reactions with random primers. According to p27gene sequences (GenBank M19499.1), PCR primers were designed asfollowed: the forward primer5'-GGA TCC ATG CCG AGA ACA TTAAAT GGG-3'and the reverse primer5'-GTC GAC TGC CAT TAA TCTAGC CTT CTG-3'. The PCR product was cloned into pMD18-T vectorand sequenced. Positive product was subcloned to pET-23a plasmid andtransformed into E. coli BL21(DE3)plysS. Target protein expression wasinduced by IPTG and purified by His resin affinity chromatography, andthen was identified by Western blotting. Then Blab/c mice wereimmunized with the purified p27protein for three times. The spleen cellswere fused with myeloma cell line SP2/0. Indirect ELISA was used to detect the titers of p27McAbs produced by hybridoma cells.Immunohistochemistry and flow cytometry were used to detect viralantigens in CEM×174cells infected with SIVmac251.
Results: p27protein was successfully expressed and purified and six hybridomacell lines stably-secreting anti-p27monoclonal antibody were obtained.Two of6kinds antibodies could be used for immunohistochemistryexperiments, and another one for flow cytometric analysis of intracellularstaining.
Conclusions: Anti-SIV p27protein monoclonal antibody was successfully prepared,which layed the foundation for the future detection of SIV infection inanimal experiments.
引文
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