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《Progress in Veterinary Medicine》 2018-05
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Construction and Identification of Ovine NYD-SP27 gene Lentiviral Vector

CHEN Yun-lei;YUAN Li-ming;LI Na;Ma Ya-ru;XU Jin-feng;SAI Wu-jia-fu;College of Animal Science and Technology,Shihezi University;College of Life Science,Shihezi University;  
In order to obtain interference of shRNA lentiviral vector of NYD-SP27 gene in ovine,in this research,the RNAi Target Sequence Selector software was used to design and synthesize 4 expressing sequences(shRNA1,shRNA2,shRNA3,shRNA4)and 1 control sequence(NC)for ovine NYD-SP27 gene,and then concatenating them into a blank plasmid vector pLentiLox3.7(PLL3.7)respectively,the constructions of 4 shRNA interfering vector NYDSP27-shRNAs and 1 negative control vector NC-shRNA were completed,meanwhile,co-transfecting interfering vector and helper plasmid into EK-293 FT cell instantaneously.the BHK-SP27-21 cells were infected with virus liquid that purified by ultrafiltration device after concentration,the relative expression of NYD-SP27 was detected by real-time quantitative PCR after 48 hinfection,and the carrier with the most efficiently interference vector was used to further experimental study.The results showed that the recombinant lentivirus vector was successfully constructed and packaged into lentivirus NYDSP27-shRNA-LV as well as BHK-SP27-21 cells were infected by recombinant lentivirus.The efficient interference ovine NYD-SP27 fragment-NYDSP27-shRNA-1 was screened by fluorescence quantitative PCR after 48 hinfection.This study laid the foundation for further research on the down-regulation of NYD-SP27 gene expression in ovine sperm development.
【Fund】: 国家自然科学基金项目(31460683)
【CateGory Index】: Q78;S826
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