The Optimization Of The Expression Of Human PapillomaVirus Type 16 L1 Gene In Pichia Pastoris GS115

Human papillomaviruses(HPVs) comprise a family of more than 130 genotypes and can be divided into cutaneous or mucosal types. Mucosal HPVs are further subdivided into either `high, intermediate' or `low-risk' types depending on the lesions they are associated with. `High-risk' types(e.g. HPV16,18) are predominately associated with cervical cancer while `low-risk' types (e.g. HPV6,11) are commonly detected in genital warts.Until now more than 100 types of HPV have been identified.The types associated with diseases of the anogenital tract can be classified on the basis of phylogenetic Relationship and of association frequencies with benign or malignant cervical lesions as low-risk types (HPV-6, -11, -34, -40, -42, -43, -44) and high-risk types (HPV-16,-18, -31, -33, -35, -39, -45, -51, -52, -54, -56, -58, -59, and -66). The most common HR types are HPV-16, -18, -31, -33, and -45.The most commonly found LR types are HPV-6 and -11.HPV are small(55-60nm),non-enveloped icosahedral DNA viruses which infect cutaneous and mucosal epithelia。It has ten open reading frame(ORF), earlyregion,late region,long control region.(1)Earlyregion include six open reading frames:E1,2,4-7。Human papillomavirus (HPV) is etiologically involved in virtually all cervical cancers, and the early .HPV oncoproteins E6 and E7 are responsible for the malignant .phenotype, mainly through inactivation of tumor suppressor proteins such as p53 and pRB. Previous work evaluating different markers of exposure and viral activity in tumors indicates that HPV may also play a role in some cancers of the oral cavity and oropharynx. Several studies have investigated prevalence of HPV in these cancers, but the prevalence of HPV detection varies broadly, depending on the population, combination of subsites, type of specimen, and detection method.(2)Late region(LR) include L1,L2. Expressed L1 or L1/L2 protein could be assembled into virus-like particles (VLPs) in vitro retaining their natural conformation and biological activity as capsid proteins. Nevertheless, for all the existing protocols of HPV16 VLPs purification, CsCl ultra-centrifugation was obligatory. This procedure is tedious and costly, thus limiting its wider application, especially in developing countries.(3) Long control region(LCR) call noncoding region(NCR),too.World- wide, cancer of the cervix is the second leading cause of cancer death in women (behind breast cancer) and is the most common form of cancer among women indeveloping countries, with an estimated 500,000 cases diagnosed each year, resulting in over 200,000 deaths annually.The L1 protein has own group in the really nuclear cell to fit out theviral type pellet the characteristic. The massive experimentsindicated that, HPV the L1 protein has stronger immunity original, canstimulate the organism to produce and the immune body, may protect theanimal in the animal experimentation to exempt the correspondingvirus's attack; Moreover, the L1 protein also has the enhancementorganism cellular immunity reply the function. The so-called viraltype pellet (VLP), is a kind becomes by certain virus' clothes protein component spontaneous assemblies Does not contain the virus genetic material, has the similar viralthree-dimensional conformation (are many is 20 hedron structures) thepellet always to call. At present studies the discovery, to VLP tookthe tumor vaccine carrier, has immunity original strong, safenon-toxic, is easy to depute and the mass productions, the vaccinationway is diverse and so on the merit, also the correlation vaccinealready obtains a better effect in some animal experimentations andthe clinical experiment.HPV16L1 VLP may stimulate the organism to produce and natural immunebody IgG, IgA, thus avoids because infecting HPV16 to cause to havethe correlation tumor possibility, it has immunity original and theimmunity comprehensive nature strong characteristic. HPV16 L1 immunityoriginal is strong, its basic reason is because it has forms VLPs theability. VLPs has greatly strengthened immunity original, can berapid, highly effective set up suddenly shape cell DC the s capture,in VLPs easy with expresses symbol the and so on MHC II kind of member,CD80, CD86 antigen presents the cell (to contain DCs, macrophage, theB cell and so on) unifies and is absorbed. HPV16L1 VLP also has thevery strong immunity comprehensive nature, it can rapidly induce DCs phaenotype and in the function is mature, the Th1 cell polarization andthe homologous T lymphocyte proliferation. Low dosage HPV16L1 VLP in does not have adjuvant under the participationcondition then to induce the organism to have the highly effectivebody fluid immunity and the cellular immunity (the CTL response).Therefore, HPV16L1 VLP both takes the preventive vaccine theapplication, and may take the treatment vaccine the carrier.The purpose of this experiment is optimizes human papilloma virus 16 L1 in Pichia Pastoris' secretion expression, lays the foundation take the time as the development preventing and controlling cervical cancer DNA vaccine. The experiment first uses the yeast to be partial codon carries on the optimization to the HPV16L1 gene, and directs the thing using PCR to extend the law design synthesis after the optimized gene, After measured the foreword expresses 16 type people papilloma virus L1 gene recombination to the secretion yeast the carrier (pPICZαB) to form expresses the virus-liked particle,phonograph yeast cell GS115 screens the multi-copies conformity clone,has obtained an expression quantity higher strain after the gene group PCR screening and rocker expression screening. Ferments the pot this strain with 5L to express and to carry on the condition to try to find out that, optimized the fermentation medium, and supplement Vc and the protein peptone reduction goal protein degeneration in the induction stage. On the cell clear the SDS-PAGE electricity swims after the ammoniumsulfate solution concentration the examination demonstration to havethe special egg unofficial receipt belt, also most is high in thefourth day expression quantity, expresses the product monomermolecular weight is about 55kD. Will ferment on the serumpurification, under the electricity mirror observes obviously VLPs.The result indicated that, was partial after the yeast codon optimizes the HPV16L1 gene in Pichia Pastoris to obtain the expression; Passes through to 5L fermentation pot expression condition trying to find out, we initially establishedstably expressed the HPV16L1 protein the craft, for further prepared the HPV VLP vaccine to lay the foundation.