Establishment of a gp120 transgenic mouse model with 7 nAChR knockout / 南方医科大学学报

OBJECTIVE@#To construct a HIV-1 gp120 transgenic mouse model (gp120) with 7 nicotinic acetylcholine receptor (7nAChR) gene knockout.@*METHODS@#The 7nAChR gene knockout mice (7R) were crossed with HIV-1gp120 transgenic mice (gp120) to generate F1 generation mice. We selected the F1 mice with the genotype of 7R/gp120 to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and 7nAChR inhibitor, and the expressions of IL-1β and TNF- were detected using ELISA.@*RESULTS@#The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (7R/gp120) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express 7 nAChR but with high gp120 protein expression. In the cell experiment, treatment with gp120 promoted the secretion of IL-1β and TNF- in BV2 cells, while inhibition of 7nAChR significantly decreased the expression of IL-1β and TNF- ( < 0.001).@*CONCLUSIONS@#By mating gp120 Tg mice with 7R mice, we obtained gp120 transgenic mice with 7nAChR gene deletion, which serve as a new animal model for exploring the role of 7nAChR in gp120-induced neurotoxicity.

Preparation and identification of anti-HIV-1 gp120 monoclonal antibody / 中国生化药物杂志

Purpose To Prepare anti- HIV-1 gp120 monoclonal antibodies and to identify the specificity of antibodies in order to provide technique for preparing HIV remedial antibodies. Methods The gene fragment of HIV-1 gp120 was connected to PEGX-4T-2 prokaryotic expressing vector. The vector was cut by enzyme. GST-HIV protein was expressed by E. coli XL1-blue. The BALB/c mice were immunized with purified GST-HIV antigen, and then the fusion of mice spleen cell and myeloma cell SP2/0 was executed as the routine cell-fusion technique. Positive cells were screened by Indirect ELISA. Immuno-blotting assay and Western blot identified the specificity of antibodies. Results External gene section from the recombinant plasmid by sequencing showed the same size of HIV-1 gp120 gene sequences. An external expressed protein band of 32 KD was obtained after purified protein SDS-PAGE electrophoresis. It indicated that six strains of hybridoma cells secreting special monoclone antibodies had been obtained. ELISA results showed that six strains monoclonal antibodies only reacted to HIV-1 gp120 antigen. Western blot results showed that a band with molecular weight 32 kDa was obtained, which could interact with HIV-1 gp120 monoclonal antibody. Conclusion Six strains of hybridoma cells secreting special monoclone antibodies had been obtained. The prepared monoclonal antibodies have established a basis for HIV remedial antibody.