Development of an immunocontraceptive DNA vaccine using sperm

我们以前报道过，主动免疫雌性大鼠rSMP-230，rSMP-B蛋白的残基1-28，和80 kDa的人精子抗原（80 kDa的HSA），防止受孕。本研究的目的是利用编码rSMP-B或80 kDa HSA的表达载体开发免疫避孕疫苗。结果如下：（1）与GenBank“808109”中报道的RSD-1编码rSMP-B的核酸序列（位置？70-773：ATAT），4个核苷酸不同（位置770-777：CACTCTG），导致3个氨基酸的差异（His-Met至Pro-Leu-Try）。(2)当将含有RSD-1的p3 xFLAG-CMV-14载体转染到HEK 293细胞中时，HEK 293细胞的细胞提取物通过使用抗FLAG抗体的Western印迹显示20-kD条带，但在培养基泳道中未发现免疫反应。(3)用80 kDa HSA合成肽主动免疫家兔，引起显著的抗体滴度。这些抗体在ELISA中特异性识别幼稚蛋白，并诱导人、大鼠和猴精子的体外凝集。这些结果表明，精子蛋白rSMP-B和80 kDa HSA是开发免疫避孕疫苗的候选蛋白。

We previously reported that active immunization of female rats with rSMP-230, residue 1-28 of rSMP-B protein, and 80 kDa human sperm antigen (80 kDa HSA), prevented conception. The purpose of this project was to develop an immunocontraceptive vaccine using an expression vector encoding rSMP-B or 80 kDa HSA. Results are as follows : (l) Compared with the nucleic acid sequence of RSD-1 encoding rSMP-B reported in GenBank"808109" (position ?70-773 : ATAT), 4 nucleotides were different (position 770-777 : CACTCTG), resulting in a difference in three amino acids (His-Met to Pro-Leu-Try). (2) When the p3xFLAG-CMV-14 vector containing RSD-1 was transfected into HEK293 cells, cell extracts of HEK293 cells showed a 20-kD band by Western blotting using an anti-FLAG antibody, but no immunoreaction was found in the lane of culture medium. (3) Active immunization of rabbits with synthetic peptides of 80 kDa HSA elicited significant antibody titers. These antibodies specifically recognized the naive protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. Western blot analysis showed that these antibodies bind to the 80 kDa band of sperm extractThese findings demonstrated that the sperm protein rSMP-B and 80 kDa HSA were pomising candidates for the development of an immunocontraceptive vaccine