关键词: ssp411, RNA干扰, 短发夹RNA, 实时定量PCR

Abstract: Objective To identify the role of new sperm-specific gene ssp411 in sperm maturation, fertilization, and embryo development, the expression vectors of ssp411 for RNA interference as siRNA-ssp411s were designed and contructed. Methods The interfering sequences of ssp411 were designed according to the sequence of ssp411 of GenBank.Three pairs of oligonucleotides were synthesized and inserted into plasmid pRNAT-U6.1 to generate siRNA expression vector, and were identified by PCR and sequence analysis. The recombinant plasmid siRNA-ssp411s and ssp411 expression plasmid ssp411-pDsRed was cotransfected into the cultured 293T cell line. The interfering efficiency was detected by real-time PCR. Results Three ssp411 siRNA frames were successfully inserted into the plasmid vector pRNAT-U6.1，and the recombinant plasmids were sent to the sequence analysis. The result of real-time PCR indicates that the siRNA expression vector which was designed by SSP411 protein coding area 3-22 got the best interfering efficiency as 71%. Conclusion The siRNA recombinant can be constructed successfully by RNAi technique for inhibiting ssp411 expression.

Key words: ssp411, RNA interference, short hairpin RNA, real-time PCR