Study On Establishment Of BHK-21 Cell Line For Stably Expressing Ovine NYD-SP27 And Selection Recombinant Lenti-virus Interference Vector

Mammalian spermatozoa could not undergo fertilization without further maturation,when some specific morphological and physiological characteristics changed,termed 'capacitation',then spermatozoa have the ability to fertilize.For natural fertilization to take place,timely capacitation and induction of the acrosome reaction were important,and maintenance of spermatozoa in an uncapacitated state prior to ejaculation was thus essential.Till now,little was known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources had been thought to be involved.PLC? plays a fundamental role in reproduction and development.The effect mechanism of phospholipase C zeta(PLC?)for egg activation and early embryo development was a hot topic in studies of reproduction and development.However,testis development specific protein gene 27(NYD-SP27)have been identified as a novel intrinsic decapacitation factor(DF)in sperm,which belonged to the PLC? family.It have confirmed NYD-SP27 played a pivotal role in regulation of sperm capacitation in mouse and human,but so far there were no reports on sheep.In this study,to provide a scientific basis for revealing the functionatin of NYD-SP27 for spermatogenesis and sperm capacitation,that ovine NYD-SP27 was cloned,expression feature of NYD-SP27 was verified and high-efficiency interference vector were constructed and selected.[Objective]In this study,to reveal the effects of over-expressing and down-regulated of NYD-SP27 on sheep spermatogenesis and sperm capacitation in the future that we supported some molecular tools,including of constructing of eukaryotic expression vector pDsRedl-C1 for NYD-SP27,Establishment of BHK-21 cell line for stably expressing ovine NYD-SP27 and construction and selection of ovine NYD-SP27 recombinant lenti-virus PLL3.7-shRNA interference vector.[Method]In order to obtain ovine NYD-SP21 gene,testicular tissue RNA was extracted and reverse transcription,then ovine NYD-SP27 was cloned and P2A-Flag-NYDSP gene was amplified,then inserted into eukaryotic expressing vector pDsRedl-C1.To co-express Red fluorescent protein gene and NYD-SP21 gene,both of them were linked with porcine teschovirus-1 2A peptide(P2A).The recombinant plasmid pDsRed-P2A-Flag-NYDSP was transfected into BHK-21 cells and selected with G418,testing Red luminescence after post-transfected by fluorescence microscope.Positive cells were identified using PCR?RT-PCR,indirect immunofluorescence assay(IFA)and Western blot.We compared morphology and growth rate between Stable cell line that cultivating 20 times of cell passages and normal BHK-21 cell.According to the ovine NYD-SP27 gene sequences,shRNA sequences and negative control sequence were designed to construct PLL3.7-NYDSP-shRNA lentiviral plasmids.All shRNA lentiviral plasmids with helper plasmids(VSVG,pMDL and REV)were co-transfected into HEK-293FT cells for packaging of the lentivirus using lip2000,respectively.Detected the Titration of packaging of the lentivirus and infected BHK-SP27-21.Rreal-time PCR was used to identify the interfere efficiency at the mRNA level of the NYD-SP27.[Results]The ovine NYD-SP27gene was amplified,and its sequences length was 1617bp by sequencing.The gene P2A and Flag tag were added in target gene.The eukaryotic expression vectors were confirmed successfully.The specific band about 1617bp were amplified by PCR and RT-PCR,suggesting NYD-SP21 gene was stably integrated into genome of BHK-21 cells.The result of IFA showed that green fluorescence was seen in positive cells,but not negative control cell.And further confirmed as the NYD-SP21 protein could be expressed in BHK-21 cell line by IFA and Western blot and the molecular weight was observed approximately 63 ku,which suggested that the self-cleavage of P2 A realized during the translation process of NYD-SP27 protein and red fluorescent protein.We finally successfully designed and constructed 4 ovine PLL3.7-NYDSP-shRNA lentiviral plasmids and found that the NYDSP-shRNA-1 was the most efficient of the four shRNAs.We observed a significant reduction in level of mRNA using qRT-PCR.[Conclusion]Finally,the ovine NYD-SP27 gene was cloned and recombinant eukaryotic expression plasmid pDsRed-P2A-Flag-NYDSP was constructed.the BHK-21 cell lines stably expressing ovine NYD-SP27 was successfully established in this study,named BHK-SP27-21 cell.NYDSP-shRNA-1 was the best shRNA for interfering ovine NYD-SP27,and could significant reduce the expression of NYD-SP27.