The Preparation And Identification Of Monoclonal Antibodies For P27Gene Of Simian Immunodeficiency Virus

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.