Molecular Identification Of Complement C3 And C5 Of Microtus Fortis And Construction Of C3 Target Recombinant Adeno-associated Virus

The adult Schistosoma japonicum parasitizes in the portal vein and mesenteric vein of its terminal host.The eggs produced by the adult worms can induce strong immune pathological reactions such as granulomas and fibrosis.Praziquantel,the only widely used large-scale therapeutic drug,is effective against adult worms,but has poor efficacy on schistosomula.The development of new drugs and vaccines and other innovative control technologies is the consensus of many parasitologists.Microtus fortis is a known mammals with strong natural resistance to Schistosoma japonicum in China.Studies have shown that their serum complement components have killing effects on schistosomula.Further research on the role of Microtus fortis complement can provide information for the prevention and treatment of schistosomiasis.In this paper,the complete gene sequences of complement C3 and C5 of Microtus fortis were cloned by RACE and nested PCR.The results showed that the ORF of the C3 gene of Microtus fortis contains 4988 bp,which encodes 1662 amino acids.The analysis results showed that the N-terminal of the C3 protein has a signal peptide composed of 24 amino acids,without a transmembrane region,and is an extremely unstable hydrophilic protein.The C5 protein is composed of 1688 amino acids encoded by a 5481bp nucleic acid,with a signal peptide composed of 25 AA at the amino end,and is a stable hydrophilic protein.The domain analysis showed that the C3 protein has 8 main domains,and the C5protein has 10 main domains.The domains of the two proteins are similar,most of them belong to the A2M family such as A2M,A2M?N,A2M?N?2 and MG1,both of them have anaphylatoxins,ANATO.Sequence alignment and phylogenetic tree analysis showed that both complement proteins C3 and C5 of Microtus fortis are the closely related to those of Microtus ochrogaster,and are relatively conservative in the evolutionary process.The expression level of complement C5 in the main organs of Microtus fortis was detected by qPCR,and the results showed that the expression of C5 was the highest in the liver.The expression level of C3 in the liver and lung of Microtus fortis increased rapidly at 1d after infection with S.japonicum cercariae,which was about 94%and 72.9%higher than that in the uninfected group.The expression level of C3 in the liver and lung was decreased at 7d and 14d after infection,and was increased or close to that in the uninfected group at 21d and 28d after infection.In the liver and lung tissues of Microtus fortis after infection,the expression level of complement C5 gene increased in the liver and lung on the 1st and 7th day after infection,and decreased on the 14th day after infection.The C5 expression level of 21d and 28d post infection in the infected group was close to that in the uninfected group.The results showed that C3 and C5 in lung and liver tissues were in a process of increase-decrease-rise(close to)with infection.The mean values of CH50 in serum of Microtus fortis on day 1,7,14,21,28 and 42 after infection with cercariae were 433U/mL,395 U/mL,378 U/mL,411U/mL,373 U/mL,555 U/mL and 433 U/mL,respectively.The results showed that there was no significant change in the serum complement activity of Microtus fortis in the course of Schistosoma japonicum infection.According to the complement C3 gene sequence of Microtus fortis,three specific ShRNA C3 were designed and inserted into an adeno-associated viruses 8(AAV8)vector to construct three recombinant viruses with a titer of more than 10¹¹ GC/ml.The rAAV8-sh RNA5-MfC3 and scramble virus can infect293T cells,as well as Hepa1-6 cells,and fluorescence signals can be observed after infection.The results indicate that the constructed recombinant virus can be expressed in liver cells.Km mice were injected with Scramble and rAAV8-sh RNA4-MfC3 virus for 4 weeks,their liver tissue had obvious fluorescence signal.Fluorescence signal in kidney of mouse injection with scramble is obvious,and is weak with rAAV8-sh RNA4-MfC3.After 3 weeks of injecting rAAV8-sh RNA4-MfC3 virus into Km mice,quantitative PCR results showed that the recombinant virus can induce liver C3 expression to be reduced by 20.03%compared with scramble,and its inhibitory efficiency reached 62.70%after 4 weeks(P<0.01).The results indicate that a recombinant adeno-associated virus that can inhibit the expression of C3 was screened preliminarily.In conclusion,the complement C3 and C5 genes of Microtus fortis were identified,and their expression patterns in the infection process were ascertained.Two recombinant adeno-associated virus that could inhibit the expression of C3 was constructed and screened,which provided a basis for in vivo test.