| Varicella-zoster virus(Varicella-zoster virus,VZV)is a type of enveloped,doublestranded DNA virus.Generally susceptible people from children to adults,from respiratory infections,into the body,induce rashes such as chickenpox or herpes zoster.VZV virus is neurotrophic,it can be latent in the nervous system,and some people cannot be completely cleared.When it is activated again,it can cause surface skin diseases such as herpes zoster and more serious nerve verification problems with ganglion conduction.The symptoms of nerve pain can last for months or years and have a great impact on the normal life of the patient.Most of the varicella and shingles vaccines on the market are attenuated vaccines.There is a certain degree of safety and possibility of recurrence.The effectiveness of the shingles vaccine in people over 50 years old is not very good.The current research on VZV vaccines is focused on recombinant vaccines.On the surface of VZV virus envelope,there are eight glycoproteins such as gE,g B,g H,g I,g K,g N,g C and g L,among which gE protein Because of its complete structure,the virus is most abundantly expressed on the surface and plays an important role in virus transmission.It is also one of the main antigens that induces humoral and cellular immunity of the host.It has become the core protein of the recombinant VZV vaccine.In this paper,the recombinant DNA of gE protein will be constructed,and the recombinant protein will be expressed using Hansenula yeast expression system and insect baculovirus expression system.The antigenicity of the two expressed proteins will be compared to compare the impact of the expression system on the production of gE recombinant vaccine.In order to compare the advantages and disadvantages of the two expression systems for the expression of gE protein,the recombinant plasmids GAP-α-gE-p WHYZ,AOX-α-gE-p WHY-Z,and GPsp-gE-p Fast Bac Dual were constructed and introduced into Hansenula respectively Expression systems and insect baculovirus expression systems.The yeast signal peptide alpha factor and viral signal peptide GPsp are used to replace the original gE signal peptide sequence,so that it can form a secreted expression.The α signal peptide-guided gE fragments were constructed under the GAP and AOX promoters of p WHY-Z vector,respectively,and the optimal expression method of yeast was found by comparing the gE proteins induced by different promoters.The gsp fragment guided by GPsp was screened by blue and white spots and transfected into sf9 cells for expression.The two expression systems were evaluated by determining the titer of the final expressed protein,comparing the amount of protein,and considering the economic cost and other factors.Recombinant plasmids were constructed and gE-Hansen yeast protein and gEinsect cell protein were expressed by two expression systems respectively,Hansen yeast fermentation culture and sf9 cell blind transmission of virus to P5 generation for antigen titer detection,the results obtained are:(1)Recombinant plasmids under two different promoters were constructed in Hansenula respectively,and it was found that the recombinant plasmid secreted by the GAP promoter secreted less protein than the secreted protein induced by the AOX promoter;(2)fermentation product detection found Although the yeast signal peptide was designed to obtain secreted expressed protein,the final result showed that the amount of intracellular expressed protein was much higher than secreted expression;(3)The antigen titer was also measured in the context of total protein,gE-Hansen yeast protein The antigen titer is 1: 102400/36 mg,and the gE-insect cell protein antigen titer is 1: 1600/36 mg.In summary,through the comparison of antigen titer determination,the expression of gE recombinant protein by Hansenula expression system is better than that of insect baculovirus expression system.In this experiment,the expression of the recombinant protein was compared with the expression of glycoprotein E of VZV in two different expression systems. |
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