| The HA gene was amplified from SIV by RT-PCR and constructed expression plasmid of HA-pCI-neo.6-8 weeks old BALB/c mice were immunized muscules with purified recombinant plasmid HA-pCI-neo. Splenocytes from the immunized mice were fused with Sp2/0-Ag-14 myeloma cellsafter the fourth immunization. Hemagglutinin (HA), hemagglutinin inhibition(HI) test and indirect ELISA was used to screen hybirdoma cells. Seven hybirdoma cell lines specific antibodies against the HA protein of H1N1 subtype SIV were established by limiting dilution assay. These were named 8C4, 8C6, 9D6, 8A4, 8B1, 9B2, 11B4. We extract total RNA in the hybridoma cell line 8C4 by PCR, reverse transcription, separated of heavy chain variable region (VH) genes, and the sequence analysis.We had 7 hybirdoma cell lines for the HA protein of H1 subtype SIV. 8C4, 8C6, 9D6 hybirdoma cell lines for the HA protein of H1 subtype SIV possess hemagglutination and neutralization activity.The HI titers of ascites of the 7 McAbs against the HA protein of H1 subtype of SIV were 512-256000, and the ELISA titers of ascites were 160000-1024000. It was shown that the immunoglobulin subclasses of 8C4, 8C6, 9D6, 9B2 were IgG2a; that of 8A4 was IgG1, that of 8B1 was IgM and 11B4 was IgG2b. Western-blot analysis confirmed that the McAbs named 8C4, 8C6, 9D6, could only react with 72KD HA protein of H1 subtype SIV. HI testion show that these 3 McAbs only reacted with SIV H1N1 and A/PR/8/34(H1N1), but did not reacted with other SIV, AIV, NDV, ADV, et al. The results of neutralization test on MDCK indicated that 8C4, 8C6, 9D6 McAbs against the HA protein of H1 subtype SIV had the neutralization capacity, and were coincided well therapy function.The McAbs bearing the HI activity had different neutralization capacity which implied that they were against the different antigen determinants in virus.This provides useful information for further studying the structure and the function of the virus.We had got VH gene from 8C4 by RT-PCR. Compared it with IMGT and BLAST data bank, VH gene contains 375 nucleotides, encoding 125 amino acids. There are 8 amino acids GYTFTSYY in CDR1, 8 amino acids IYPGDGST in CDR2, and 9 amino acids ARVMIAMDY in CDR3. The 22 and 96 cysteine emerge the mark of disulfide bond in the VH gene. The heavy chain by VH, DH and JH 3 gene fragments encoding all kinds of heavy chain constant region gene-based together at the lower reaches of the variable region genes. The DH gene encodes the most of CDR3 in V region of heavy chain, JH gene ligates with V and C gene, and encodes the V region CDR3 and FW4 except DH encoding region. Because the variable region is encoding with several fragments, and each fragment exists as a group genes. So that the gene fragmens have to link up to form a complete function variable region gene by gene rearrangemengt. The V gene coming from 8C4 derived from IgHV1S56*01, the J gene derived from IgHV1S56*01, and the D gene may derive from IgHD6-1*01 or IgHD6-1*02 or IGHD6-2*01 or IgHD6-2*02 or IgHD6-3*01. V, D, J genes by post-rearrangement linked together form a V-D-J complex, completed heavy chain rearrangement. The sequence of mouse Ig heavy chain in line with the basic framework of the structure, the framework of the region without stop codon is generated by rearrangement sequence. 8C4-VH1 with the report number IGHV1S56*01 sequence homology rate of 96.88%, but antibodies IGHV1S56*01 sequence n did not refer more at CDR3 hypervariable regio. Obtained two light chain gene sequence analysis, we can see that the antibody light chain are pseudogenes.These McAbs can be used as a useful tool to analyze the antigenic variation. They also provide the effective reagents for the rapid detection of subtype H1 of SIV. The heavy chain sequence laid the foundation material for the development of single domain antibodies and single-chain antibody in this experiment. |
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