Development of an immunocontraceptive DNA vaccine using sperm

我们之前报道过，用rSMP-230、rSMP-B蛋白残基1-28和80 kDa人精子抗原（80 kDa HSA）主动免疫雌性大鼠，可以防止受孕。本项目的目的是利用rSMP-B或80 kDa HSA的表达载体开发一种免疫避孕疫苗。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（His-Met到Pro-Leu-Try）的差异。(2)将含有RSD-1的p3xFLAG-CMV-14载体转染到HEK293细胞后，用抗flag抗体进行Western blotting， HEK293细胞提取物显示出20-kD条带，但在培养基中未发现免疫反应。(3)用80 kDa HSA合成肽主动免疫兔，可获得显著的抗体滴度。这些抗体在ELISA中特异性识别该蛋白，并诱导人、大鼠和猴子精子的体外凝集。Western blot分析表明，这些抗体与精子提取物的80 kDa带结合，表明精子蛋白rSMP-B和80 kDa的HSA是开发免疫避孕疫苗的理想候选物

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