Abstract
Objective: HepG2.215 cells are used as an experimental model, the central interaction region (CID) of MxA protein is divided into different regions according to its structure, and different plasmids are constructed to find the most powerful functional polypeptide against hepatitis B virus. Methods: Using full-length MxA plasmid as a template and pCMV-tag-3A as a vector, we constructed plasmids A1, A1A2, and A2A3 according to the CID. The plasmids were sequenced and transfected into HepG2.2.15 cells respectively. 24 hours later, the expression of the plasmid was detected by qRT-PCR. And the expression of HBsAg and HBeAg were detected by enzyme-linked immunosorbent assay (ELISA). The relative DNA expression of HBV DNA was detected by qRT-PCR. Combined with the peptide structure and the screening results, the segment was again divided into different short peptides, which were constructed into different plasmids and transfected into cells. And we detected the corresponding indicators again. Thereby repeat in this cycle until the shortest functional polypeptide is found. Result: The constructed plasmids were correct and their expression was normal. The expression of HBV DNA, HBsAg and HBeAg in the A1 group were significantly lower than that in the other two groups, and it was equivalent to the CID group. Separating the A1 component into A1N, A1C, the results of the test showed that the expression levels of HBV DNA, HBsAg and HBeAg in the two groups were higher than those in the A1 group and the CID group, and there was no difference between the two groups and the blank group.
Highlights
Chronic hepatitis B virus (HBV) infection is closely related to liver diseases[1]
The results show that HBV interference with JAK1-STAT1 signaling pathway and antagonistic effect against viral protein is an important mechanism to cause IFN-α's http://www.ijSciences.com
HBV core antigen HBcAg can be combined with the promoter region of the myxovirus resistance protein A (MxA) gene to reduce the expression of MxA protein[9]
Summary
Chronic hepatitis B virus (HBV) infection is closely related to liver diseases (such as chronic liver dysfunction, cirrhosis and liver cancer)[1]. The MxA gene is normally silent, when the cells are infected by virus or are highly expressed induced by type I interferon, they produce two direct antiviral factors 2'-5' oligoadenylate synthase 1 (OAS-1) and myxovirus resistance protein A (MxA). Since it is discovered, MxA protein has attracted much attention due to its broad-spectrum anti-RNA virus activity. 1.3Total RNA extraction and real-time PCR The total RNA was extracted by Takara Rnaiso plus after cell treatment, and the reverse transcription was carried out using transcript one-step gDNA Removal and cDNA Synthesis supermix Reverse Transcription kit, with a reaction condition of 42°C15min , 85°C5s, the synthesized cDNA is preserved in -80°C. The significant level difference was set as P < 0.05
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