The Study Of Enhancement On Human Papillomavirus Major Capsid Protein Solubel Expression In Escherichia Coli

Human papillomavirus(HPV), a non-enveloped doublestranded DNA virus, can infect normal tissues and cause tumors of the skin and mucosa. HPV infection could causes benign or malignant lesions of skin, mouth, tongue, tonsils, throat, genital which included flat warts, genital warts and other malignant lesions include head and neck cancer, throat cancer, cervical cancer and anal cancer. According to the World Health Organization, there are 160 million clinical cases of cervical cancer in worldwide in 2012. There are 528,000 new cases and cause 26.6 million deaths in the same year, while there are 130 000 new cases per year in China and keep in continued growth trend. According to cervical intraepithelial neoplasia(CIN) grade which lead by HPV, the operation such as electrocautery, laser, microwave, freezer, cervical loop electrosurgical excision are used for removal the affected part and combine with a variety of antiviral drugs such as recombinant human interferon, aciclovir, interleukin for further consolidating the therapeutic effect. But diseases which caused by HPV is difficult to effectively control with the higher recurrence rate which difficult to completely cure. The severe cases will increase the probability of occurrence of malignant tumors. Therefore, prevention of high-risk HPV infection become the focus. The most important tools is prophylactic vaccine. The people who get vaccinated will obtain capability that resist HPV infection.Due to the HPV neutralizing epitopes primarily exist on the surface of the major capsid protein L1, therefore HPV virus-like particles(VLPs) is as a platform for the development of a preventive vaccine. So far, there are three licensed VLPs vaccines by the US Food and Drug Administration(FDA). Although, HPV prophylactic vaccines have no significant treatment effect of cervical cancer. Its subtypes restriction and high prices limit promotion and popularity especially in developing countries with high incidence of cervical cancer. However, it is the most effective drug for prevent high-risk HPV infection. Therefore, reducing restrictions subtype and reduce the cost effectively is an urgent problem.In first chapter, we have described consist of HPV, its life cycle, induced disease, pathogenesis, means of screening, treatment, prevention methods and limitations and also described function, mechanism of the heat shock protein as well as in protein folding and importance of soluble expression. Moreover we have described the crystal structure of HPV L1 and stability and solubility of L1 pentamers which impact by helix 5. After that, the thesis and significance is raised.In second chapter, we have constructed the coexpression system in order to solve soluble problems of HPV major capsid protein expression in E. coli. In this section, major capsid protein fusion with GST and coexpressed with GroEL/ES, Dna K/DnaJ/GrpE or TF E. coli respectively. Through the study of overexpression in E. coli, GroEL/ES is a major factor in the increase of soluble GST-L1 expression. With the enhance of soluble GST-L1 expression, yield of L1 pentamer also will be significantly improved. Overexpression of GroEL/ES effectively improve the cell growth rate. In the co-expression system, the high overexpression of GroEL/ES could bind a lot of GST-L1 that affects purification and the yield of L1 pentamer. ATP and MgCl2 introduced in the purification process to promote the release of GST-L1 and to enhance binding efficiency with affinity chromatography. Moreover promoted the L1 pentamer yield. Through the optimization of expression and purification conditions, there was 5, 3 and 2 fold enhancement in pentamer yield compared with the control for HPV16, 18 and 58 L1 respectively. HPV L1 expression improvement in E. coli provide a new method for optimizing the HPV prophylactic vaccine production process.In third chapter, based on mutations of Leu on 469 site(L469) of HPV16 L1 α helix 5(helix5: h5) enhance the yield of soluble L1 pentamer. The stability of L1 associated with at this site, therefore, the site of 469 were mutated to Ala(A), Gly(G), Ser(S), Lys(K) and observed the changes in L1 yield of soluble pentamer. To explore the reason of the improved yield of L1-p, NAMD and NACCESS were applied to L469 A as an example. After MD simulation, we found that the secondary structure of h5, βG2, βB1, βC, βD and βF have changed. This change contributed to improvement on the hydrophobic interaction with β core “ jelly ” and stability of h5. In addition, it induced h5 moved away from core structure and exposed the carboxyl terminal residues on the surface of L1. This change improved the negative charge on surface of h5 which contributed to stability and solubility of L1. On the other hand, the mutation affected the SASA of L469 A, especially in the section of h5. The decrease of surface area of A469 will reduce hydrophobic surface with solution. Moreover, the improvement on SASA of residues of h5 which located on surface will improve the stability of L1 in hydrophilic environment that profit for formation of L1-p.