Construction And Identification Of Recombinant BCG Vaccine Of IL-2 And Epstein-barr Virus LMP1 Fusion Gene

Objective In this study,LMP1 was fused with human IL-2 gene and linked to pMV261 plasmid to construct a recombinant plasmid.Then importing the constructed recombinant plasmid into competent BCG(BCG)by electroporated to obtain recombinant BCG(rBCG)that capable of stably expressing the fusion gene.Detection of immune effect of rBCG by inhibiting tumor growth in constructed Epstein-Barr virus positive nasopharyngeal carcinoma animal model.The rBCG laid the foundation for a bivalent vaccine that against EBV-positive tumors and anti-tuberculosis.Method Total RNA was extracted from Epstein-Barr virus-positive B95-8 cells.Using total RNA as a template to obtain cDNA by RT-PCR.Then,LMP1 was obtained by PCR with the cDNA template.IL-2 was obtained by PCR,and the template was pMalLP plasmid which contain the IL-2 target gene.The IL-2 gene and LMP1 gene were fused by overlap-PCR.The fusion gene and plasmid pMV261 were digested with restriction enzyme,and ligated the fusion gene to the plasmid by T4 DNA ligase to obtain the recombinant plasmid.Then,the obtained recombinant plasmid was identified by enzyme digestion,PCR and sequencing.Transfering the correctly identified recombinant plasmid into competent BCG by electroporation.Promoting the expression of fusion genes by temperature induction.Extracting the total protein and detecting the expression of fusion gene by western-blot.Immunized EB virus positive nasopharyngeal carcinoma animal model with the obtained rBCG.Regularly measured the long diameter and short diameter of tumor tissue and calculated its volume.Then,sacrificed the experimental animals,weighed the tumor tissues and calculated the tumor tissue inhibition rate.Observed the inflammatory cell infiltration by HE staining.Result The total RNA extracted from B95-8 cells was detected by agarose gel electrophoresis,the results showed that the band was clear and the total RNA was available.The full-length cDNA of LMP1 was obtained by RT-PCR.To obtain 1225 bp LMP1 with the designed specific primer by PCR.Amplification to obtain 453 bp IL-2 with plasmid that containing the target gene as the template.Fusing of IL-2 and LMP1 by overlapping PCR to obtain 1623 bp fusion gene.Then,the fusion gene was ligated into the plasmid pMV261 and the recombinant plasmid was identified by enzyme digestion,PCR and sequencing.The about 1600 bp fragment was obtained by PCR amplification and the fragment size is as expected.Two fragments of 1600 bp and 4500 bp were obtained by restriction enzyme digestion,and the size was consistent with the expected.The sequencing result showed that the fusion gene sequence was 100% identical to the known sequence.The constructed plasmid was electroporated into BCG competent cells.The fusion protein that encoded by fusion gene in rBCG can be detected by western-blot.There was no significant difference in tumor formation time and tumor volume between rBCG group,BCG group and PBS in animal experiments.At the same time after immunization,the tumor volume of the rBCG group was smaller than PBS group and BCG group,and the BCG group was smaller than the PBS group.The average tumor weight of the rBCG group was smaller than that of the BCG group and the PBS group.There was no significant difference between the BCG group and the PBS group.The tumor inhibition rate of the rBCG group was 38.00% higher than the BCG group(11.91%).Conclusion The recombinant plasmid pMV261-IL-2-LMP1 was constructed and it was successfully introduced into BCG to obtain rBCG which can stably express the fusion protein.The constructed rBCG has an inhibitory effect on the growth of EB virus-positive nasopharyngeal carcinoma.