| Synaptonemal complex (SC) is an endonuclear protein complex that is formed during the homologous chromosome matching in the first meiosis prophase in eukaryotic germ cells. SC proteins (SCPs) are temporarily expressed during leptotene of the meiosis prophase, and they are degraded during diplotene. SCPs are evolutionarily conserved in most mammalian species and SC has been reported to be involved in chromosome condensation, recombination and double-strand breaks (DSB), homologous chromosome matching and a number of other important bioprocesses. Synaptonemal complex (SC) proteins, especially SCP3, play a crucial role in SC formation during male germ cell meiosis.It has been not got the sequence of the Cashmere goat Scp3 gene up to now. In this study, we cloned the Scp3 gene of Cashmere goat, and examined Scp3 transcription in prepubertal Cashmere goat testes. Finally we prepared the polyclonal antigen gene protein. And it is good basis for the next preparation of the polyclonal antibody. Firstly, based on the analysis of homology of the Scp3 sequence among the cattle, human, mouse and rat, different sets of primers were designed to amplify the corresponding unknown region of cashmere goat. We successfully cloned the coding sequence of Cashmere goat Scp3 gene. Our result suggested that the PCR products are around 850 bp, cloned Scp3 matched the predicted size of Scp3 gene. The PCR products were ligased into pMD-19T vetor and sequenced. When the Scp3 gene sequence derived from Cashmere goat was compared with those from rat, mouse, human and cow, data suggested that they were highly homologous (> 83%), especially between goat and cow Scp3 genes (homology > 98%).In addition, we investigated the development of prepubertal Cashmere goat tests by using molecular biological approach. After the Scp3 coding sequence of Cashmere goat was identified, we analyzed the Scp3 mRNA expression in testicular tissues of Cashmere goats at different time points, and discovered the time for first round meiosis. The goat testicular tissues from postnatal days 45, 56, 66, 69, 73, 86, 95,102, 110, and 122 were analyzed for Scp3 transcriptional expression. The tissues from postnatal days 56 and 73 to 122 were detected for Scp3 transcription, whereas the tissues from postnatal days 45, 66 and 69 were not detectable. These results illustrate that the first round meiosis in the Cashmere goat testes occurs mostly behind postnatal day 73. Therefore, we could not define a specific time point for the first round meiosis in goat testis, though the first round of meiosis in analyzed Cashmere goat testes occurred mostly behind 73 days after birth.Based on the coding region sequence, we prepared polyclonal antigen of SCP3. Firstly, we analyzed epitope sequence and protein structure of the Scp3,and to identify high-epitope fragment for PCR amplification. the amplified fragment and prokaryotic expression vector pET-44a (+) were ligased. The recombinant protein pET-44a-SCP3 was induced in 1mmol / L of IPTG, 6 hours. When expressed the protein, we purified them via affinity chromatography. After identification of PCR and restriction enzyme digestion, sequencing of positive clones, the sequencing results with known sequences are the same. And the results showed that pET-44a -SCP3 were successful constructed. At the same time, SDS-PAGE, Western blot assay of protein expression. Around a 15kDa protein band with the expected molecular weight, since the end of the recombinant protein with 6 His tag fusion, to recognize His antibody, anti-His antibodies and purification for the fusion protein after Western blot hybridization, the same color after the emergence of a band of 15kDa that the protein is purified with a His tag fusion protein. At last, all the results showed that we have successfully got the right SCP3 antigenic protein. |
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