摘要
为取得大鼠睾丸存在γ－氨基丁酸（ＧＡＢＡ）样受体的直接分子生物学证据，根据神经组织ＧＡＢＡ受体α，β，γ和ρ亚基多肽链第二跨膜区保守８肽的ｃＤＮＡ序列设计合成４条寡核苷酸探针，经地高辛（ＤＩＧ１１ｄｄＵＴＰ）３’末端标记后与成年大鼠脑和睾丸ＲＮＡ进行斑点（ｄｏｔ）杂交分析，结果显示大鼠睾丸较其大脑有较强的杂交信号，表明睾丸组织的ＧＡＢＡ受体ＲＮＡ比脑组织ＧＡＢＡ受体ＲＮＡ与探针序列有较多的同源区段，提示该受体可能是一种类似于ＧＡＢＡ的受体，即ＧＡＢＡ样受体（ＧＡＢＡｒｅｃｅｐｔｏｒｌｉｋｅ）。基于上述研究结果，进一步杂交筛选大鼠睾丸ｃＤＮＡ文库，经两轮杂交筛选，共获得４３个杂交阳性噬菌斑克隆。为进一步鉴定这些克隆，以上述探针为５’端引物，另合成Ｔ７启动子互补序列为３’端引物；经聚合酶链反应（ＰＣＲ）对上述克隆ＤＮＡ进行扩增，凝胶电泳显示其中２２个分别出现０９ｋｂ或０６ｋｂ的特异扩增带，表明它们可能是含有大量睾丸ＧＡＢＡ样受体ｃＤＮＡ的阳性克隆。
In order to take the direct evidence of molecular biology whether GABA receptorlike is in rattestis.24mer 4 oligonucleotides to be complementary to the conservative region(8 peptides)of the cDNA of neurGABA receptor were synthesized and labeled with DIG11ddUTP,then the oligonucleotides were used as probes to hybrid respectively wity the RNAs of rat brain and testis.The result of the dot hybridization showed out that the hybrid signal of rat testis was stronger than that of rat brain,with the meaning that, in comparing with rat brain,the RNA of rat tastis had more identical regions with probes,and also implying that,in the structure and biological function,the GABA receptor from rat testis RNA might be different from,but similar to neurGABA receptor.Following the above research,the λ ZAPⅡ cDNA library of rat testis was screened by repeating hybrid twice,as a positive result,the 43 plaques were selected from the library.For making clear further,if they contained the cDNA of GABA receptor,using oligonucleotide probes above as upper primer,the complete complementary sequence of T7 promoter which was located 3’ end of cloning site as lower primer,then 22 clones of 09kb and 06kb fragments which were amplified via PCR were identified.
出处
《华西医学》
CAS
1998年第3期369-372,共4页
West China Medical Journal
基金
国家攀登计划
美国洛氏基金
中国科学院上海细胞生物学研究所分子细胞生物学开放实验室资助
关键词
顶体反应
Γ氨基丁酸
受体
CDNA
克隆
睾丸
γaminobutyric acids receptorlike
cDNA clone
Dot hybridization
PCR.
