Objective: To express the protein of HPV18E6 based on pET-32a(+) at high level and study the expression and significance of HPV18E6 proteins in laryngeal carcinoma. Methods: The HPV18E6 gene was amplified by PCR and cloned into pET-32a(+). The amplified fragment was inserted into the plasmid pET32a (+) that was digested with BamHⅠand Hind Ⅲ. The recombinant plasmid pET32/E6 was transformed into E.coli JM109 which was selected with ampicillin. The recombinant plasmids were successfully introduced into E.coli BL21(DE 3) and were induced by IPTG. SDS-PAGE and Western blot analysis were used to detect the confusion protein. Finally, the optimization of expression conditions, such as temperature, concentration of IPTG, was studied. Results: The recombinant plasmids were identified and confirmed with enzyme digestion and sequencing. The BL21(DE3) transformed recombinant plasmid pET32/E6 had expressed HPV18E6 recombinant protein effectively. The optimum conditions of expression were 37 ℃, 1 mmol/L IPTG. Conclusion:Prokaryotic expression vector pET-32a(+)-HPV18E6 was successfully constructed. The high-level expression of HPV18E6 was achieved in E.coli BL21(DE3).

Prokaryotic expression vector of mouse HPV16E6 gene was constructed. A pair of primers were designed according to the digestion sites in plasmid pGEX-KG and the HPV16E6 gene sequence published by GenBank. The DNA fragment of 321bp was amplified by PCR from the HPV recombinant plasmid with HPV16E6 gene, then cloned into pGEX-KG and transformed into the host E. coli strain JM109. The fragment was conformed to the original sequence, which indicated that fusion expression vector pGEX-KG-HPV16E6 was constructed. The pGEX-KG-HPV16E6 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degrees C, the expression product of HPV16E6 gene was identified by SDS-PAGE and Western blot. HPV16E6 fusion protein had been expressed successfully in the form of inclusion bodies, the molecular weight of fusion protein being 38 kD. Meanwhile, the optimum condition of HPV16E6 fusion protein expression was induced with 1.0 mmol/L IPTG for 4h. The fusion protein reacted specifically with the antibodies against HPV16E6. HPV16E6 gene was successfully expressed in E. coli, which could be used as a basis for preparing HPV16E6 vaccine in human.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Repressor Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Objective: To evaluate the efficacy and safety of laparoscopic surgery in the treatment of gallstones and common bile duct stones. Methods: Eighty-seven patients with gallstones complicated with common bile duct stones who underwent concurrent laparoscopic surgery at Zhoushan Hospital from December 2015 to December 2017 were enrolled.The patients were divided into A group and B group according to the digital table.A group(38 cases) underwent laparoscopic cholecystectomy (LC) combined with laparoscopic common bile duct exploration (LCBDE), and B group(49 cases) underwent endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy (EST) combined with laparoscopic cholecystectomy (LC). The curative effect of the two groups was observed.The operation time, the success rate of the operation and the rate of laparotomy were recorded in the two groups.The corresponding hospitalization time and cost were compared.The safety of the two different procedures was compared after surgery, and the complications of the two groups were recorded. Results: In A group, the average diameter of common bile duct stones was (1.02±0.25)cm, the average diameter of common bile duct diameter was (1.15±0.25)cm.In B group, the mean diameter of common bile duct stones was (0.99±0.26)cm, and the average diameter of common bile duct was (1.13±0.26) cm.The differences between the two groups were not statistically significant (t=0.513, 0.437, 0.367, P=2.083, 1.533, 1.095). The successful operation rate of A group was 92.11%(35/38), which in B group was 91.84%(45/49), the difference was not statistically significant between the two groups(χ²=0.006, P=0.974). The incidence rate of complications in B group was 20.41%, which was significantly higher than that in A group, the difference was statistically significant(χ²=3.654, P=0.019). The hospitalization time, hospitalization expenses in A group were (10.6±2.6)d, (26 649.8±3 478.6)CNY, respectively, which were significantly better than those in B group (t=21.971, 17.168, all P<0.05). Conclusion The efficacy of LC combined with LCBDE for patients with gallstones complicated with common bile duct stones is better than ERCP/EST combined with LC surgery, and the safety of the former is higher than the latter.