HLA-A33 Expression In Vitro And Identification

The Human Leukocyte Antigen (HLA) system is an important genetic material of mankind which is the most complex diversity system known so far. HLA-A33 epitope is a common clinical epitope which account for 21% of the total HLA-A allele among Chinese. According to latest report, HLA-A33 plays a crucial role during the progress of many diseases-the association with Viral Hepatitis chronicity recorded by statistical result of South China and Korean; a sensitive gene of EV71 by Doc. Luan-Yin Chang's research; the genetic correlation to Acute Lymphoblastic Leukemia (ALL) reported by Jun Hong etc..This study aimed to grip the techniques of high efficiency and specific expression of HLA-A33 protein in both prokaryotic expression system and eukaryotic expression system, which include but not limited to construction, identification, transfection and transform of recombinant prokaryotic and eukaryotic expression vectors, array of protein expression level. Furthermore, we do some preliminary study on the interaction protein of EV71 and HLA-A33 which will offer theory basis and experimental guide for pathological observation and immune mechanism.This study includes 2 experiments.Experiments 1: Establishment of a cell line with stable expression of HLA-A33 protein and research on its function. Optimization the codon of HLA-A33 gene inquired at GenBank according to prokaryotic expression rules and design necessary restriction enzyme cut sites Nhe I , Xho I and Flag-tagged gene. Integrate the recombinant gene with host cell line genome using lentivirus system. Infect the cell line with EV71 and then a CoIP assay is performed to search the interaction protein.Experiments 2: Eukaryotic expression and identification of HLA-A33 protein. Optimization the codon of HLA-A33 gene inquired at GenBank according to eukaryotic expression rules and design necessary restriction enzyme cut sites Nco I and BamH I. Construct a recombinant vector by T4 ligastion by using pET28a as host vector. Transform targeted DNA into TOP10 competent cell with the method of chemical transformation to get enough copies. Identify the new clone by PCR and sequencing. Re-transform the DNA into BL21 (DE3) to express HLA-A33 protein. This protein has BSP and Streptavidin which enables the implement of the following research on the construction HLA-A33 tetramer.The above empirical study comes to the following conclusions: Successfully constructed eukaryotic expression vector with common molecular cloning technique and stable expression cell line of HLA-A33 with lentivirus system and do some preliminary study on the interaction protein of EV71 and HLA-A33 which will offer favorable theory basis and experimental guide for further research on the function of HLA-A33.