重组人钙调素的表达及其McAb的制备与精子CaM定位

EXPRESSION OF RECOMBINANT HUMAN CALMODUL IN Ⅲ AND PRODUCTION OF ITS MONOCLONAL ANTIBODY AND LOCALIZATION IN HUMAN SPERM

用基因重组技术构建人钙调素基因Ⅲ（ｈＣａＭⅢｃＤＮＡ）表达载体ｐＢＶ／ｈＣａＭⅢ，并在大肠杆菌ＤＨ５α中经热诱导获得可溶性ＣａＭ蛋白的高效表达．将纯化的重组ｈＣａＭ与异型双功能剂ＳＰＤＰ及鼠血清白蛋白（ＭＳＡ）交联，免疫Ｂａｌｂ／ｃ小鼠，用常规细胞融合技术制备单克隆抗体（ＭｃＡｂ），得到３株稳定分泌单克隆抗体的杂交瘤细胞．间接ＥＬＩＳＡ和斑点免疫结果证实，三种单克隆抗体均与自制的ｒｈＣａＭ和ＣａＭ标准品起特异反应．用免疫组化法对精子中ＣａＭ定位，发现ＣａＭ主要分布于精子头颈部，不育组ＣａＭ＋精子率（（４５．０±７．５）％）显著低于生育组（（６８．５±１０．５）％）．

The human calmodulin (hCaM) cDNA recombinant expression vector (pBV200/hCam) was constructed by gene recombinant technique. The recombinant plasmid was transformed into E. coli DH5α. After heat induction, a high level expression of soluble CaM was obtained. The purified rhCaM, crosslinked with SPDP and MSA, immunized Balb/c mice. After cell fusion and screening, three hybridoma cell strains secreting monoclonal antibodies (McAbs) constantly was generated, and the specificity of the McAbs was demonstrated by indirect ELISA and dot immunoblot. Localization of CaM in human sperm was detected by immunohistochemical method.