HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.
Animals ; Capsid Proteins ; biosynthesis ; Codon ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Human papillomavirus 16 ; Mice ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; biosynthesis ; Papillomavirus E7 Proteins ; biosynthesis ; Papillomavirus Vaccines ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis

Objective    To evaluate the strategy of chemoradiotherapy following endoscopic R0 resection for esophageal cancer in M3-T1b stage. Methods    There were 45 esophageal cancer patients with M3-T1b stage with endoscopic R0 resection followed by additional chemoradiotherapy from ECETC (Esophageal Cancer Endoscopic Therapy Consortium) as a trial group with 34 males and 11 females at age of 61.37±7.14 years. There were 90 patients with esophagectomy from Fudan University Shanghai Cancer Center as a control group with 63 males and 27 females at age of 61.04±8.17 years. Propensity score match (1:2) was used to balance the factors: gender, age, position, depth of invasion and lymphovascular invasion (LVI), which may influence the outcomes. Overall survival (OS) rate, relapse free survival (RFS) rate, and local recurrence rate were compared between the two groups. Result    There was no statistical difference (HR=2.66 with 95%CI 0.87 to 8.11, P=0.179) in terms of OS rate between the two groups. One, two and three years overall survival rate of patients in the control group was 93%, 86%, and 84%, respectively. Nobody died in the trial group within 3 years after surgery. The RFS rate between the two groups didn’t significantly differ (HR=1.48, 95% CI 0.66 to 3.33, P=0.389). One, two and three years RFS rate of patients in the contorl group was 87%, 78%, and 76%, respectively, while 97%, 93%, and 73% in the trial group, respectively. The local recurrence rates between the two groups didn’t significantly differ either ( HR=0.53，95%CI 0.13 to 2.18, P=0.314). One, two and three years local recurrence rate of patients in the control group was 5%, 6% and 6%, respectively, while 0%, 0% and 21% in the trial group, respectively. Conclusion    Similar outcomes are found regarding OS, RFS and local recurrence rates between the two groups. The strategy of endoscopic R0 resection followed by additional chemoradiotherapy has prospect for the treatment of esophageal cancer in M3-T1b stage. And this kind of therapy may be provided for those with risk factors or can not tolerate surgery.