Construction of the human sperm acrosomal membrane-associated protein SAMP32 DNA contraceptive vaccine

摘要

Objective: To establish the DNA contraceptive vaccine of hSAMP32. Methods: The hSAMP32 DNA was acquired by RT-PCR based on the hSAMP32 sequence in Genbank, and subcloned into pGEM-T-vector; and the sequence was detected after digestion. Then the pcDNAS.1 ( + ) -hSAMP32 plasmid DNA was constructed and quantified. The pcDNA3.1(+)-hSAMP32 DNA was transfected into NIH3T3 cells by Lipofectin. The hSAMP32 mRNA expression was detected by in-situ hybridization. Results:(1)The distincttotal RNA bands corresponding to 28 S and 18 S clearly were isolated from human testis tissue. The length of amplified RT-PCR product was consistant with that of proposed target gene, hSAMP32. (2)The sequence of RT-PCR product linked with pGEM-T vector was co-incident with that of hSAMP32 in Genbank. (3) The supercoiled pcDNA3.1 ( + ) -hSAMP32 contained no endotoxin. (4) The pcDNA3.1(+)-hSAMP32 DNA in ultra-helix pattern was double times higher than that in linearized form, and the endotoxin was negative. (5)The positive NIH3T3 cells were selected by 400mg/L G418. (6) The sensitivity of the labled probe approached up 0.1pg. (7) The bluish-violet colored granules were located in cytoplasm, indicating that hSAMP32 gene could express in NIH3T3 cells. Conclusion: The pcDNA3.1(+)-hSAMP32 DNA could be efficiently transfected into NIH3T3 cells by Lipofectin-mediated gene transfection technique, and it's expression could be detected by in-situ hybridization. Establishment of DNA immuno-vaccine pcDNA3.1 ( + ) -hSAMP32 by using subcloning technique may provide experimental based for research of contraceptive vaccine.