DNA vaccine for preventing Japanese encephalitis and its construction method
A DNA vaccine and Japanese encephalitis technology, applied in the field of DNA vaccines, can solve the problems of immune response type biased towards humoral immunity, short duration of immune efficacy, high price, etc., to avoid immune attenuation, long-lasting immunity, and safety high effect
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Embodiment 1
[0026] Example 1 Construction of DNA vaccine pVAX1-SA14ME eukaryotic expression plasmid for Japanese encephalitis
[0027] 1. At first carry out the preparation of the target gene of antigen coding: with the original JEV SA14-14-2 strain shown in SEQ ID No.2 prM / E The gene is optimized as a template, and the optimization is mainly aimed at the partial tropism of the human species and the reasonable reduction of GC content in the sequence. The original sequence is synonymous with the amino acid sequence translated into the optimized sequence, but the nucleotide sequences of the original sequence and the optimized sequence are quite different. After optimization, the optimized gene shown in SEQ ID No.3 was obtained prM / E , the gene is artificially synthesized, and the artificial synthesis method is a known technique. optimizing genes prM / E Before inserting SEQ ID No.4 shown wxya The signal sequence gets wxya Signal sequence optimization prM / E Gene. On the basis of the s...
Embodiment 2
[0030] Example 2 In vivo and in vitro expression of the Japanese encephalitis DNA vaccine pVAX1-SA14ME constructed by the present invention
[0031] In order to verify the immunogenicity of the plasmid pVAX1-SA14ME constructed in Example 1, it is necessary to verify the target gene in vitro and in vivo after ensuring that the sequencing is completely matched. To this end, we transfected the plasmid pVAX1-SA14ME or the pVAX1 empty vector of the present invention into BHK-21 cells. After 48 hours, mouse JEV antiserum was used as the primary antibody, and FITC-labeled goat anti-mouse antibody was used as the secondary antibody. Indirect immunofluorescence staining ( figure 1 AB); the plasmid pVAX1-SA14ME or pVAX1 empty plasmid was immunized into the leg muscles of mice, and one week later, the muscles at the immunized site were sliced, the mouse JEV antiserum was used as the primary antibody, and the HRP-labeled goat anti-mouse antibody was used as the secondary antibody. chemi...
Embodiment 3
[0033] Example 3 Immunogenicity evaluation and protective effect evaluation of Japanese encephalitis DNA vaccine pVAX1-SA14ME constructed in the present invention
[0034] 1. In order to ensure that pVAX1-SA14ME can be used for subsequent applications, its immunogenicity and protective effect need to be verified, because the immune protection induced by JEV is mainly based on humoral immunity. ) mice for IgG titer and neutralizing antibody titer (PRNT 50 ), and challenged the mice after immunization to preliminarily evaluate its protective effect. Specific steps are as follows:
[0035] The challenge test was carried out on the mice after immunization (intraperitoneal injection of 1×10 7 PFU Beijing-1 strain of JEV virus, destroying the blood-brain barrier). figure 2 A is the dynamic anti-JEV IgG titer; figure 2 B is the dynamic anti-JEV neutralizing antibody titer; figure 2 C is the dynamic change of body weight after challenge; figure 2 D is the survival rate; fi...
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