Protein Expression Of Human TCP11a And ZNF313 Genes In E. Coli And Immunohistochemical Studies On The Expression In Mouse And Human Testicular Tissues

Spermatogenesis is generally divided into three different phase:(1) a proliferative phase characterized by rapid mitotic divisions of spermatogonia, (2) a meiotic phase in which spermatocytes changed into spermatids after reduction divisions with chromosome pairing and crossing over, (3) spermiogenetic phase in which spermatids transform into spermmatozoa. These process require highly regulated expression of many tesit-specific and non-specific genes.In recent years the Department of Medical Genetics, Huaxi Hospital of Sichuan University has devoted itself to the cloning and function study of the genes related to human male sterility.Four human and two mouse spermatogenesis-related genes, have been cloned by mRNA differential disply, namely ZNF230, ZNF463, ZNF313, TCP11, Znf230 and Znf313. These new genes are highly expressed in testiclar tissues, and were suggested to be crucial in human reproductive ability. Further functional studies are undergoing withyeast double hybridization, gene knock-out and RNA interference techniques.Part I: Protein expression of human TCP11a gene and its distributions on cells and tissueHuman TCP 11 gene encodes huamn receptor fertilization promotive peptide. It has several splicing variants. One of the variants, TCPlla, was used to prepare its protein, antibody and for study on the protein distribution of testicular tissue and cells.First, the TCPlla gene was successfully cloned to pBV220 through PCR technology, and was verified by DNA sequencing and N-terminal analysis. By the optimizing condition, TCPlla protein was highly expressed in E. coli. Then we prepared the antibody from the purified protein and investigated the distribution of the expressed protein in the testicular tissue and cells by immunohistochemistry method.The result revealed that the TCP11 protein was mainly located in the cytoplasm and membrane of prolonged spermtid rather than spermatogonium, spermatocyte, Sertoli cell and Ledying cell. This suggested that the TCP11 gene expresses at the later stage of germ cell maturation, and might be important for the morphological and physiological changes during the spermatogenesis. Further study indicated that the TCPlla protein was distributed on the acrosome and the tail of the matured spermtids, which was coherent with the resultsof the distribution of mouse TCP11 protein.Bioinformatic analysis of TCP11 gene in rat, mouse, chicken and chimpanze, including the homology and conservatism of the exon - intron structure revealed that many conserved non-coding sequences existed in about 30kb upstream of TCP11, which might be the enhancer or other regulatory elements for the specific expression of TCP11 genes. Intensive studies of the cis-elements on one of the most conserved non-coding sequence revealed that the latter possible contains many binding sites for regulation factors. Besides, a CpG island around the promoter of human TCP11 gene was also found, in rat and mouse. The methylation and de-mythylation of the CpG island may be involved in the transcription regulation of TCP11. The predictive studies of the secondary structure of TCP11a protein revealed that only helix presented in the secondary structure, and there existed many post-transcription modification sites, especially potential the phosphorylation sites.Part II: New human spermatogenesis related gene ZNF313 : its expression in Escherichia coli and distribution on the testis tissueZinc finger proteins are a large family of DNA binding proteins which are ubiquitous in animals, plants and human beings. With their unique properties, they played pivotal role in gene expression and cell differentiation. C2H2 and C3HC4 are two basic domains of zinc finger protein, but rarely co-existed in the same protein, whereasthey do in ZNF313 protein.In order to study the gene expression the human ZNF313 gene was cloned into pET-32(a) vector with PCR technology, and the fusion protein was obtained. After was confirmed its correct expression by DNA sequencing and N-terminal analysis, further optimization of the condition for fe...