The Construction And Identification Of The Adenovirus Vector With MyD88SiRNA Of Mucaca Mulatta

[Object]To construct the adenovirus vector with MyD88siRNA of mucaca mulatta and package it.[Methods]1. The preparation of the adenovirus vector with MyD88siRNA of mucaca mulatta:firstly the target of siRNA was designed,then restriction of virus vector was completed after Oligo annealing. We go on connecting the vector of double enzyme linearization to DNA fragment for coupled reaction.The product was changed over to the E.coli TOP10competent cell. Then PCR identification were made, positive cloning sequencing were done and plasmid were extracted.2. The packaging of MyD88siRNA sequence of mucaca mulatta:HEK293cells were fostered and DNA solution was prepared.The recombinant adenovirus were got after the transfection of plasmid. We amplified the adenovirus twice then the virus were purified with Adeno-XTM purification kit.3. Adenovirus titer were measured with End-point dilution.4. The inhibitory effect of MYD88siRNA for the expression of MYD88in DC was detected by western blot.:CD34+cells were cultured for7days and induced into DC. the adenovirus vector with MyD88was infected after48hours.Whether the gene can inhibit the expression of MyD88was conformed with Western blot.[Result]After PCR amplification and electrophoresis detection,we could get the result that the positive cloning PCR segment was about343bp and the empty carrier cloning PCR segment was306bp.It was in line with the theoretical value.The result of sequencing had proved a coherence about the MyD88-RNAi sequence of mucaca mulatta and the target gene in our experiment.There was no mutation or frame shift. The result confirmed the product of PCR amplification was the target gene what we need.It indicated that we structured the RNA interference recombinant plasmids successfully.The Western blot showed that the specific bands sizes were accorded with the theory value. Because of the MyD88siRNA,the expression of MyD88in interference group was decreased significantly. It concluded that MyD88siRNA can inhibit the expression of MyD88in DC.[Conclution]Adenovirus vector with MyD88siRNA of mucaca mulatta was constrcted successfully,and the foundation of the experiment was established for using them to infect the immature dendritic cells and studying the liver transplant tolerance of mucaca multta further.