A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then ...A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.展开更多
文摘A 550 bp cDNA fragment of HSD I coding for an extracellular domain of human sperm membrane protein (hSMP 1) was ligated with an Adapter containing the universal stop codon, and the ligated fragment cDNA was then cloned into the MAS of pUC19. The desired plasmid with correct open reading frame was obtained, and was cut with EcoR I.The insert was purified and then cloned into the two asd + Salmonella expression vectors (the low copy number plasmid pYA292 and the high copy number plasmid pYA3137). The recombinant plasmid containing the insert with the correct orientation was selected by restriction enzyme digestion analysis. The recombinant plasmids were transferred into the non pathogenic Salmonella typhimurium χ4550, which was deletion of the Δcya, Δcrp and Δasd genes. Western blot analysis of the whole cell lysate of the two recombinants of S. typhimurium showed a predominant protein band at 21 KD, which reacted with the anti hSMP 1 antiserum. The result indicated that two recombinants of S. typhimurium containing the 550 bp cDNA of HSD I were constructed and the characteristics of their growth in vitro were determined. They may be used as new potential mucosal immunization antifertility.
基金The present study was supported by grants from the Rockefeller Foundation,USA,and from the National High-Tech Program for Biotechnology and 'Climb' Program of of the State Science and Technology Commission,China
