Cloning And Expression Of S100A12Gene In The Reproductive Organs Of Yak (Cornu Bovis Grunniens)

Calgnanulin C (S100A12) is a calcium binding protein of the S100family which strongly expressed by granulocytes, keratinocytes, and lower expressed by lymphocytes and monocytes. S100A12is high correlation with inflammation, and it can be secreted into inflammatory site specificity. This study was expected to exploring the S100A12gene play roles in genital system of yak by cloning the S100A12gene of yak and anglicizing the characteristic of it’s tissue specific expression.1. S100A12gene of yak was amplified by RT-PCR and then cloned into pMD19-T simple vector for sequence analysis. It was confirmed that pMD19-S100A12was built successfully by PCR and sequencing. The result of DNA sequence analysis with DNAStar and Blast on line demonstrated that the cDNA sequence contains an open reading frame (ORF) of279bp which shares the highest identity of100%with the S100A12gene of Bos taurus(Accession number:NM174651). Cladogram analysis result showed that S100A12gene of yak had a nearer genetic relationship with Sus scrofa, Equus caballus and so on.2. The gene expression profiles of S100A12gene in spleen and lymph nodes, mmammary gland and genital system (including testis, epididymis, penis, ovaries, corpus uteri, cervix, oviducts, horn of uterus and vagina) of yaks were detected by application of semi-quantity RT-PCR. The results showed that S100A12gene was strongly expressed in spleen; higher expressed in testis, epididymis, mammary gland, ovarie.3. Rabbits were immunized with the S100A12fusion protein which expressed by prokaryotic to obtain the polyclonal antisera. The titer was then tested using agar diffusion and indirect enzyme-linked immunosorbent assay (ELISA), and the specificity was tested by western blot. The titer of the S100A12was1:8as determined by agar diffusion and over1:409600by ELISA and it could specifically recognize S100A12protein by western blot. The results indicated that the high titer and fine specificity of anti-S100A12polyclonal antibody was obtained and it could be a novel tool in future investigation of S100A12functions.4. The expression level of S100A12gene in tissue samples of yaks were tested by immunohistochemistry. Average optical density was measured by computator image analysis software in different tissues. And those samples were dyed to HE at the same time to insure that the tissues were integrality. The results of the immunohistochemistry indicated that the S100A12protein was expressed strongly in spleen and lymph nodes, higher expression level in testis, epididymis, mammary gland and ovaries, and relatively lower expression in corpus uteri, cervix, horn of uterus, vagina and penis relatively.5. According to the results of this study, it is concluded that S100A12was palying an important roles in innate immune of mammary gland and genital system of yak.