Genetic engineering subunit vaccine capable of resisting goat hydatidosis infection
A technology encoding genes and amino acids, which is applied in the field of animal immunological drugs, can solve the problems of low glycosylation level, easy formation of inclusion bodies, and poor folding type in the expression system of Pichia pastoris, and achieve strong solubility, good immune effect, and high expression level. high effect
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[0064] In some embodiments, a method for preparing a genetically engineered subunit vaccine against echinococcosis infection specifically includes:
[0065] 1) cloning the eukaryotic expression vector comprising the optimized Eg95 protein coding gene;
[0066] 2) Transfect CHO cells, and obtain a CHO cell line that stably and efficiently expresses Eg95 protein in suspension through selection and screening;
[0067] 3) The cell strain screened out in step 2) of fermentation and cultivation is purified to obtain recombinant Eg95 protein;
[0068] 4) The recombinant Eg95 protein is thoroughly mixed, and then fully mixed with an adjuvant to obtain a recombinant expression subunit vaccine.
[0069] The method provided in the above embodiments of the present invention can harvest the target protein from the cell culture supernatant, and the yield is as high as 2-3g / L, which not only shortens the protein purification time and simplifies the vaccine production steps, but also greatly...
Embodiment 1
[0082] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-Eg95-GS
[0083] 1. Amplification and purification of Eg95 gene The codon-optimized Eg95 gene (SEQ ID NO: 1) was synthesized in Shanghai Sunny Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-Eg95 plasmid vector. PCR amplification was performed using pUC-Eg95 as a template and Eg95-F and Eg95-R as primers (the gene sequences of Eg95-F and Eg95-R are shown in SEQ ID NO: 3 and 4). The amplification system is shown in Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.
[0084] Table 1 Eg95 gene amplification system
[0085]
[0086] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, a band appeared near the po...
Embodiment 2
[0098] Example 2 Construction and Screening of Recombinant CHO Cells Expressing Eg95 Protein
[0099] 1. Cell Transfection
[0100] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.
[0101] 1.2 Plasmid and cell mixing Take 5 μg of the pCI-Eg95-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.
[0102] 1.3 Electroporation 280V 20ms electric shock for 2 pulses. Immediately after the electric shock is completed, the cells are transferred to a shaker fl...
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