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

ObjectiveTo evaluate the efficacy of different dones of urapidil in preventing pituitrin-induced cardiovascular responses in patients undergoing laparoscopic myomectomy.MethodsSixty ASA Ⅰ or Ⅱ patients,aged 27-41 yr,weighing 55-65 kg,scheduled for elective laparoscopic myomectomy under general anesthesia,were randonly divided into 4 groups (n =15 each):control group (group C) and urapidil 0.3,0.5 and 0.8 mg/kg groups (groups U1-3).Urapidil 0.3,0.5 and 0.8 mg/kg were injected intravenously in U1-3 groups respectively,while normal saline 5 ml was given in group C.The mixture of pituitrin 6 U and normal saline 20 ml was injected into the site of hysteromyoma 5 min later.The operation was then started.BIS value was maintained at 45-55.The occurrence of cardiovascular responses was recorded.ResultsThe incidences of cardiovascular responses were 100％,67％,40％ and 20％ in groups C and U1-3 respectively.The incidence of cardiovascular responses was significantly lower in groups U1-3 than in group C,and in groups U2.3 than in group U1 ( P ＜ 0.01 ).There was no significant difference in the incidence of cordiovascular responses between U2 and U3 groups (P ＞ 0.05).ConclusionUrapidil can prevent pituitrin-induced cardiovascular responses in patients undergoing laparoscopic myomectomy and the optimal dose is 0.5 mg/kg.