Method for preparing bacterial ghost
A technology of bacteria and bacteria shadows, applied in the field of genetic engineering vaccines, to achieve good immune protection effect
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Embodiment 1
[0033] Example 1 Construction of pET-28a-INP recombinant plasmid
[0034] First, synthesize the IN sequence according to the sequence of SEQ ID NO: 1, then use the synthesized INP sequence as a template to amplify the INP gene by PCR, and introduce NdeI and XhoI restriction sites at the 5' end respectively. The primer sequences of the INP gene are: INP-F: 5'GCG CATATGGCACTGGAT-3' (SEQ ID NO: 4), INP-R: 5'GCT CTCGAG GGTCTGCAGATT-3' (SEQ ID NO: 5), the underline is the introduced restriction site. PCR reaction conditions: 95°C for 5min, 94°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycles, 72°C for 7min.
[0035] Then, pET-28a was double-digested with NdeI and XhoI, and the INP PCR product was also double-digested, mixed with the pET-28a digested product, and then transformed into E. coli with T4 ligase in a water bath at 16°C for 30 minutes. DH5α competent cells were then plated onto Kana-resistant LB plates. The next day, the single clone on the plate was picked for PCR a...
Embodiment 2
[0036] Example 2 Construction of pMD-28a-INP recombinant plasmid
[0037] The region containing the promoter region and the INP sequence on the recombinant plasmid pET-28a-INP was used with primers pET--28a-inp-F: 5'-ATACCGCGGATCCGGATATAGTTCC-3'(SEQ ID NO: 6) and pET--28a-inp -R: 5'-AATGAGCTCTCACTGCCCGCTTT-3' (SEQ ID NO: 7) was PCR amplified. The PCR reaction conditions were: 95°C for 5 minutes, 30 cycles at 94°C for 30s, 52°C for 30s, and 72°C for 40s, and 72°C for 10 minutes. The PCR product was then cloned into the pMD-19T (simple) plasmid vector, transformed into Escherichia coli DH5α competent cells, and spread on an ampicillin-resistant LB plate. On the next day, single clones on the plate were picked for PCR and enzyme digestion identification, and the identified correct clone was named pMD-28a-INP recombinant plasmid.
Embodiment 3
[0038] Example 3 Construction of pMD-28a-INP-cap recombinant plasmid
[0039] Synthesize the cap gene sequence according to the sequence of SEQ ID NO: 3, use it as a template, and use primers cap-F: 5'-ACACATATGACGTATCCAAGGAGGC-3' (SEQ ID NO: 8) and cap-R: 5'-ACTCTCGAGTTAGGGTTTAAGTGGG-3' (SEQ ID NO: 9) PCR amplification of the cap gene was performed. The PCR reaction conditions were: 95°C for 5min, 94°C for 30s, 52°C for 30s, 72°C for 10s, 30 cycles, 72°C for 10min. Then use Nde I and Xho I to double digest the pMD-28a-INP and cap gene PCR products, and use T4 ligase to infect the digested products in a water bath at 16°C for 30 minutes, then transform E. coli DH5α competent cells, and then apply to ampicillin resistant LB plates. On the next day, single clones on the plate were picked for PCR and enzyme digestion identification, and the identified correct clone was named pMD-28a-INP-Cap vector plasmid.
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