Study on the expression and function of Epstein-barr virus vIL-10 gene
Objective The prokaryotic expression vector of Epstein-Barr virus vIL-10 gene was constructed and vIL-10 was purified.Mouse experiments were conducted to explore its effect on MC38 tumor cells,laying the foundation for further exploring the role and mechanism of vIL-10 in the body.Methods According to the DNA sequence of vIL-10,primers were designed and the vIL-10 gene was obtained by PCR amplification;recombinant plasmid vIL-10-pGEX-4T-1 was constructed and transformed into E.coli.DH5α;positive colonies were screened on resistance plates,and plasmids were extracted and then subjected to PCR,double enzyme digestion and sequencing identification;the correctly identified recombinant plasmid vIL-10-pGEX-4T-1 was transformed into E.coil.DE3 and screened with ampicillin,induced by IPTG for expression,and the bacteria were broken by ultrasonic wave to extract protein and identified by SDS-PAGE and Western blot;The positive strains were cultured in large quantities,and the proteins were extracted after the bacteria were broken.The vIL-10 was purified using the GST tag,and a small amount of the purified product was used for SDS-PAGE and Western blot identification again;the GST tag was cut off by thrombin,and the protein content was determined after desalting and concentrating the protein in the dialysis bag for later use.MC38 cells were collected and subcutaneously injected into 6-week-old wild-type and IL-10 knockout C57BL/6J mice of similar weight.Three weeks later,mice with successfully constructed tumor models were injected with vIL-10 at 400 μg/kg peritumoral infiltration,once every 7 days,for a total of 2 injections.Mice were killed 7 days after the last injection,and the tumors were removed,weighed,recorded and analyzed.Results The size of the vIL-10 gene obtained by PCR was about 513 bp.The results of PCR and double enzyme digestion of the recombinant plasmid vIL-10-pGEX-4T-1 were in line with expectations,and the gene sequencing results were consistent with the vIL-10 template sequence.E.coil.DE3 transformed with vIL-10-pGEX-4T-1 was induced to express by IPTG.SDS-PAGE electrophoresis showed that a 45 ku band was visible compared with the control group,and Western blot showed that the protein was successfully expressed.The positive strain was cultured in large quantities,and the protein was extracted and purified with a GST tag.Western blot results showed that the protein could be specifically purified.After cutting,dialysis and concentration,the protein content was determined to be 0.56 mg/ml.Animal experimental results showed that the tumor weight of wild-type(C57BL/6J)mice injected with vIL-10 protein was significantly increased compared with that of the non-injected group(P＜0.05);the tumor weight of IL-10 gene knockout(IL-10-KO)mice injected with vIL-10 was significantly increased compared with that of the non-injected group(P＜0.001).Conclusion Western blot analysis showed that E.coil.DE3 containing the recombinant plasmid vIL-10-pGEX-4T-1 could successfully express vIL-10,and animal experiments showed that vIL-10 could promote tumor growth,laying a foundation for studying the effects of vIL-10 on tumors and its mechanism.
Epstein-Barr virus(EBV)vIL-10prokaryotic expressionGST tag purification
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