人精子有效抗原基因表达克隆的研究

Study on genes encoding effective human sperm antigens

我们从人睾丸组织中提取ＲＮＡ，并进一步纯化出ｍＲＮＡ；以此为模板，在反转录酶和ＤＮＡ多聚酶的作用下，合成ｃＤＮＡ；将ｃＤＮＡ与λｇＩＩ载体重组后转染大肠杆菌，构建人睾丸ｃＤＮＡ表达文库。运用遗传显色法和噬菌斑原位杂交法鉴定表达文库后，用兔抗人精子抗体筛选人精子抗原基因表达克隆（ＨＳＧ）。对ＨＳＧ表达抗原（ＨＳＧＡｇ）和特异抗体（ＨＳＧＡｂ）进行纯化和抗生育效应的测定。结果显示：（１）人睾丸ｃＤＮＡ表达文库容量为１．８２Ｘｌ０６ｐｆｕ，遗传显色法示重组率为６７％，噬菌斑原位杂交法示重组率为５１％。（２）经兔抗人精子抗体筛选２Ｘｌ０４ｐｆｕ，得８株人精子抗原基因表达克隆。（３）人血清、兔血清ＨＳＧ２Ａｂ和兔血清ＨＳＧ８Ａｂ对人精子具有补体依赖细胞毒作用。（４）兔血清ＨＳＧ３Ａｂ能阻断人精子在顶体反应时顶体后区ＣｏｎＡ受体的暴露。结果提示，ＨＳＧ２、ＨＳＧ３和ＨＳＧ８克隆的表达抗原是精子有效抗原，能作为精子免疫避孕疫苗的候选成分。

Objectives :To investigate effective human sperm antigens for contraceptive vaccines. Design :Experimental molecular biology study. settings :Histology and embryology laboratories in medical universities.MethOds :mRNA was isolated from total RNA in human testis. cDNA was synthesized by using AMVreverse transcriptase and DNA polymerase with mRNA as template. cDNA was recombined withλgtII vec-tors and with Y1090 as infected bacteria。 Human testis cDNA library was constructed. After the library wasidentified using genetic and plaque lift hybridization,cloning human sperm antigen genes ( HSG)werescreened with rabbit's anti-human sperm antibodies. HSG expression sperm antigens( HSGAg)and corre-sponding antibodies (HSGAb) were isolated and their biological activity was determined. Results(1)In human testis cDNA library 1. 82X 106pfu of recombinantλgtII,were obtained. Geneticmethod showed that recombinant rate was 67%, plaque lift hybridization was 51%. (2)among 2 X 104pfu ofrecombinant λgtII 8 positive clones( named 1～8 ) were found which wereλgtII recombined with humansperm antigen genes.(3)Human and rabbit anti-booies of the expression products of HSG2 and HSG8 couldinhibit the motility of human sperm. (4) In the course of acrosome reaction,the exposure of Con A recep-tors on the surface of sperm was inhibited by the rabbit antibody derived from HSG3. ConclusiOn : The expression products of HSG2, HSG3,and HSG8 could be used as candidates for prepa-ration of sperm vaccines.