Construction Of RNAi Slow Virus Vector Of B2m Gene And Its Effect On Proliferation Of Hepatoma Cells

Objective: To structure the lentiviral vector of B2 m gene and investigate the effect of B2 m knockdown on the proliferation of hepatoma cells.Methods:(1)Through referring to B2 m sequence,we obtained the screened RNAi sequence by software.The shRNA sequence meeting the requirements was devised on the basis of RNAi sequence.The resulting sequence was further linked to produced double-stranded DNA.Wait until the both ends of vector was cut,we bound it to the DNA.By means of identification,sequencing and extraction,the B2 m gene RNAi lentivirus was obtained.The titer of the virus was detected by q PCR.(2)Huh-7 cells were divided into negative control virus infection group(group NC)and B2 mRNAi lentivirus infection group(group KD).The 95489-1 infection group was labeled KD1,the 95490-1 infection group was labeled KD2,the95491-1 infection group was labeled KD3.The transfection efficiency was observed under Celigo.(3)The mRNA expression of Huh-7 cells after B2 m gene knockdown was measured by real-time PCR.By evaluating the knockout efficiency of B2 m gene,effective RNAi vectors were screened.(4)Hepatoma cells infected by the selected LV-B2m-RNAi were used as group B,and the negative control virus infected hepatoma cells were used as group A,we recorded the hyperplasia amount of hepatoma cells every day using Celigo,and analyze its growth and proliferation.Results:(1)According to the designed three RNAi sequences,three kinds of B2 mRNAi lentivirus with slow titers of 2E + 9,2E + 9 and8 E + 8 were successfully constructed.(2)The LV-B2m-RNAi was used to infect hepatoma cell line(Huh-7),and the infection rate was more than80%.(3)In contrasted with group NC,the mRNA expression level of B2 m gene in group KD was obviously restrained(p < 0.05),and 93.1% of B2 m gene was knocked down successfully in group KD1.(4)Celigo counting results showed that the proliferation of group B was restrained contrasted with group A(p < 0.05).Conclusions:(1)The qualified RNAi lentiviral vector of B2 m gene and the B2 m knockout hepatoma cells(Huh-7)were obtained.(2)Knockdown the B2 m gene could restrain HCC cells(Huh-7)proliferation.