HPV16 Virus-like Particles In Insect Cell Expression System Of Optimization

HPV for most women,is a transient infection,about 80% of the female life at least once HPV infection,before it caused abnormal cells through the body’s own immune system will completely remove it,no harm to health,can not be detected according to the results of the study show that,for the first time since the discovery of HPV in cervical carcinoma,confirmed about 15 high-risk genotypes of HPV persistent infection can cause cervical cancer,precancerous lesions and 3 cervical intraepithelial neoplasia(CIN3)and adenocarcinoma in situ(AIS).In recent years,some studies found that HPV may and other cancers as well as correlation,throat cancer,for example,often occur in men,people often think before its associated with smoking,drinking,chewing betel nut,but now it has evidence to prove that,about 30-40% of the occurrence of certain type of throat cancer associated with HPV.In the HPV positive of throat cancer in the crowd,with the rapid development of modern genetic engineering technology,has set up a lot of kinds of heterologous protein expression system.For now,the expression of prophylactic HPV vaccine system including 1 e.coli expression system,2 yeast expression system,insect cell expression system.Insect cell baculovirus expression system since the 1980 s,has been continuously improved,is an important part of the expression of recombinant proteins.Compared with mammalian cell expression system,insect cell expression quantity higher,cultivating way more diverse,and better security,it has a wider application.This paper studies on the stability of the known insect cell expression system,the different virus infection of the virus the plural(MOI),different cell density,the mutual contact and influence between different cell harvest time.HPV16 type VLP build express: synthesis type 16 genes by PCR,according to the approved a signal design PCR primers amplification truncated type HPV16 type,gene sequencing right after the reorganization into the shuttle plasmid p Fast Bac1 baculovirus expression system,through genetic shift function will aim gene recombinant Bacmid genome,extract the restructuring Bacmid DNA,M13 primerPCR identification is correct,the recombinant baculovirus Bacmid-HPVL1 transfection sf9 cells infected after the insect cells of P1,P1 continue to infect cells get P2 and so on,get a lot of stable titer of poisonous kind of concentrate.And determine the poison of the MOI.Experiment 1: suspension culture SF9 cells,the cell growth under the condition of normal growth curve drawing,with certain viruses MOI values against poison every point on the growth curve,post-harvest western blot appraisal,look at that point,the expression of the highest amount.Experiment 2: fixed a point on the growth curve,with different virus attack MOI poisoned this,after harvest western blot,behold a MOI expression quantity is best,then purified by column chromatography,and finally through the antibody neutralization experiment to test whether there is immunogenicity.The experimental results show that the MOI,must be represented in SF9 cells after 72 hours of the attack poison to express the highest amount.In SF9 cells on the growth curve of fixed 72 hours after the wedding,when the virus MOI is 0.5,the expression of poison attack the highest amount.This article through to HPV the preliminary research on the production process,biological products for the future has certain guiding significance for industrial production of amplification.