Preparation Of ZIKA Virus Key Immunogen Protein And Preliminary Study Of DNA Vaccine

ZIKA virus is a mosquito-borne flavivirus.Since 2007,several large-scale outbreaks of ZIKA virus infection have occurred worldwide.Although most patients have milder symptoms of virus infection,some cases still prove ZIKA virus infection is associated with fetal microcephaly and Guillain-Barre syndrome,and has become a public health event of international concern.There is currently no effective treatment for ZIKA virus infection and no vaccine for clinical use.Therefore,the strategy of dealing with ZIKA virus infection is mainly focused on preventing infection,and it is particularly important to research a safe and effective vaccine at this time.Envelope glycoprotein,E protein,is one of the three structural proteins of ZIKA virus.Its biological function is the main component involved in receptor binding,membrane fusion,and host immune recognition.The E protein is often used as a targeting protein when developing protective antibodies against ZIKA virus infection.The purpose of this experiment is to express and purify the ZIKA virus E protein,and use specific antibodies to detect whether the protein is immunogenic.The E protein membrane outer region(E80)gene was amplified and cloned into the prokaryotic expression vector p ET-28 a.The prokaryotic recombinant plasmid p ET-28a-ZIKA-E80 was successfully constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.After IPTG induction,the protein was expressed in the form of inclusion bodies in the precipitation.After denaturing the inclusion body with 8M urea,the denatured protein was purified by Ni2+ affinity chromatography column to obtain high purity recombinant protein,and then the denaturant was slowly removed by concentration gradient dialysis to renaturata the protein.The purified recombinant protein was used as an antigen,and a specific antibody against E protein was used as a primary antibody.Western-blot detection was performed,and the results showed that the purified protein was immunogenic.Membrane precursor protein,pr M/M protein,is another structural protein of ZIKA virus.Currently known biological function is to prevent premature fusion of E protein during early processing and transportation,and to help E protein fold to form homodimeric in the later stage.The pr M/M protein is often used as an antibody-activated epitopes during the development of the ZIKA virus vaccine.In this experiment,the appropriate M protein coding sequence was selected for prokaryotic plasmid construction,and the expressed ZIKA virus M protein was purified.The entire M protein gene was first amplified and integrated into the prokaryotic expression vector p ET-28 a.The prokaryotic recombinant plasmid p ET-28a-ZIKA-M was successfully constructed,but it was found that this recombinant plasmid could not express the M protein in E.coli cell.After in-fusion clone by deleting the sequence encoding the transmembrane domain of the M protein,the recombinant plasmid p ET-28a-ZIKA-M90 was successfully obtained and transformed into competent cells of E.coli BL21(DE3).The recombinant protein is expressed in the form of inclusion bodies in the precipitate.Pure ZIKA virus M90 protein was finally obtained by the same purification method as E protein.In this experiment,the eukaryotic recombinant plasmid p CMV-S-ZIKA_ME containing the sequence encoding the ZIKA virus pr M/ E protein was first transfected into mouse muscle tissues.The muscle tissues were used as protein samples,and specific antibodies against E protein were used as primary antibodies.Western-blot analysis showed that the recombinant plasmid p CMV-S-ZIKA_ME was able to express the antigen protein pr M/E protein in muscle cells.Then use PEI as an immune adjuvant.The plasmid p CMV-S-ZIKA_ME was immunized into the mice by aerosolization.After three immunizations,the mice's eysballs were removed to collect blood samples,and the purified pr M/E protein was used as the antigen.The mouse serum was used as an antibody for Western-blot reaction,and the final results showed that the mouse serum had specific antibodies.The results indicate that the expression of the recombinant plasmid p CMV-S-ZIKA_ME can cause immunity in vivo.