Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.
Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.
复制标题
编码人顶体内抗原 SP-10 的 cDNA 的克隆和测序。
DOI：
10.1095/biolreprod42.4.693
复制
发表时间：
1990
影响因子：
3.6
通讯作者：
Herr,JC
中科院分区：
文献类型：
--
作者：
Wright,RM;John,E;Klotz,K;Flickinger,CJ;Herr,JC
编码顶体内蛋白SP-10的cDNA被克隆，并被鉴定为了解该抗原在精子发生过程中的表达的第一步。从人睾丸基因表达文库中分离到三个重叠的SP-10特异性cDNA。这些cDNA与1.35kb的mRNA杂交，这种mRNA存在于人的睾丸中，但在肝脏或胎盘中没有发现。这些DNA的完整序列命名为SP-10-5、SP-10-8和SP-10-10，产生了一个1117bp的序列，其中包含SP-10蛋白的265个氨基酸编码区。从推导的氨基酸序列产生的疏水性图显示了信号肽的一个非常疏水的氨基末端。序列数据显示，在SP-10蛋白的中央1/3处共出现了3个不同的氨基酸重复序列，共16次。有趣的是，cDNASP-10-10有一个内部57个碱基对(19个氨基酸)的框内缺失，这在SP-10-5中不存在，这表明选择性剪接产生不止一个SP-10 mRNA。SP-10蛋白似乎是一种独特的顶体蛋白，根据之前的免疫组织学数据和观察到的SP-10 cDNA序列与Genbank、国家生物医学研究基金会或瑞士序列库中发现的其他序列没有任何显著的同源性。在大肠杆菌表达载体中表达重组SP-10融合蛋白，并用于制备多克隆抗血清。这种抗血清对顶体帽进行原位染色，并与一组类似于SP-10的单抗在蛋白质印迹上反应的多肽反应。
cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10–5, SP-10–8, and SP-10–10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10–10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10–5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.