Multi-T cell epitope tuberculosis gene vaccine using hsp65 as epitope scaffold
A gene vaccine and gene technology, applied in gene therapy, genetic engineering, plant genetic improvement, etc., can solve problems such as poor prevention and treatment of tuberculosis
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Embodiment 1
[0187] Embodiment 1: Construction of pcDNA3-PES tuberculosis gene vaccine
[0188] By consulting relevant literature, five T cell epitopes derived from Mycobacterium tuberculosis H37Rv strain were selected: the 1-60 gene of ESAT-6 protein (EAST-6 1-60 ); the 721-765 gene of Ag85B protein (Ag85B 721-765 ); 7-33 gene of MTB protein (MTB10.4 7-33 ); the 721-765 gene of PPE25 protein (PPE25 721-765 ); the 10-54 gene of PE19 protein (PE19 10-54 ). Such as figure 1 As shown, using pcDNA3.1 or pVAX as the plasmid vector and HSP65 gene as the chimeric epitope gene vector, based on the computer prediction of its secondary structure, the position that does not affect the key structure: the 438th base After inserting the 1-60th gene of ESAT-6, after the 453rd base, insert the 721-765th gene of Ag85B, after the 471st base, insert the 7th-33rd gene of MTB10.4, after the 1185th base The 721-765 gene of PPE25 is inserted into the 10-54 gene of PE19, and the 7-33 gene of MTB10.4 protein...
Embodiment 2
[0325] Example 2 Construction of pET32a-HSP65 prokaryotic expression vector and protein expression and purification
[0326] In order to obtain a large amount of HSP65 protein antigen, the amplified HSP65 coding gene was double-digested with EcoR I and Hind III, and connected with the corresponding prokaryotic expression vector pET32a to construct the pET32a-HSP65 prokaryotic expression plasmid.
[0327]Escherichia coli BL21 (DE3) competent cells were transformed with pET32a-HSP65, cultured overnight at 37°C, and positive clones were screened. Shake culture in LB liquid medium until A600 reaches 0.6-1.0, and add isopropylthiosemiglucoside (IPTG) to a final concentration of 1mM. Continue shaking culture for 4 hours, collect bacteria by centrifugation at 4000r / min for 20min, resuspend in 1×PBS, sonicate, centrifuge at 12000r / min at 4°C for 20min, and harvest supernatant and precipitate respectively. The supernatant was passed through an affinity chromatography column and eluted...
Embodiment 3
[0328] Example 3 Preparation of chitosan-pES tuberculosis gene vaccine
[0329] 1) Mass preparation of plasmid DNA: according to QIAGEN Plasmid Mega Kit.
[0330] 2) Prepare chitosan solution and pcDNA3.1-PES solution respectively, the method is as follows: first prepare 0.02% chitosan solution with pH 5.7, weigh 0.02g chitosan (Fluka, MW about 400000, deacetylation degree 75%-85%) , first dissolve it slowly with 500μl-1ml 1% HAc solution, and place it at 37°C for 1hr. Then weigh 0.042g NaAc, with 80ml H 2 O is dissolved, add the above-mentioned completely dissolved chitosan solution, mix well, and adjust the pH value to 5.7 with 1N NaOH, which is 0.02% chitosan solution with pH 5.7. pcDNA3.1-PES plasmid with 5mM Na 2 SO 4 Dissolve, DNA concentration is 1mg / ml, -20 0 C save it for later use.
[0331] 3) Prepare chitosan-DNA nanoparticles by co-precipitation method (co-aggregation method), as follows: in a 55°C water bath, drop 0.02% chitosan solution with pH 5.7 into 800...
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