Recombinant human interferon beta la gene, expression vector thereof and preparation method of recombinant human interferon beta la
A technology of recombinant human interferon and expression vector, applied in the field of genetic engineering, can solve the problem of low activity of human interferon β protein, achieve high recovery rate, optimize cell culture conditions, and achieve good purification effect
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Embodiment 1-4
[0051] Embodiment 1-4 describes the construction material of engineering plasmid psv2-dhfr-IFN-β1a
[0052] Strain: Escherichia coli JM109, purchased from Shanghai Chaoyan Biotechnology Co., Ltd.; vector pUC57, Nanjing GenScript Biotechnology Co., Ltd.; psv2-dhfr was purchased from ATCC.
[0053] Main reagents:
[0054] Restriction enzymes EcoR I and Bam HI were purchased from Toyobo Biotechnology Co., Ltd. Taq DNA polymerase, Pfu DNA polymerase, ultrapure dNTPs, DNA Marker DL2000, and λHind III were purchased from Beijing Dingguo Biotechnology Co., Ltd. Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Ampicillin was purchased from Sigma. Competent cells, a large number of plasmid extraction kits, DNA purification and recovery kits, and alkaline phosphatase were purchased from Takara Biotech.
[0055] Agarose electrophoresis buffer 50xTAE: 242g Tris, 57.1ml glacial acetic acid, 18.6g EDTA. EB solution: Dissolve 1g ethidium bromide in 100ml sterile wate...
Embodiment 1
[0061] Example 1 PCR amplification of interferon beta 1a gene
[0062] 1. Obtaining the target gene:
[0063] The complete cDNA sequence of interferon β1a was retrieved through NCBI genebank on the Internet. In order to improve the stability of the protein after expression, the present invention modified the 17th cysteine of interferon β1a to obtain recombinant human interferon β1a gene , the nucleotide sequence of which is shown in SEQ ID NO.1.
[0064] The recombinant interferon β1a gene was cloned into the vector pUC57, and the recipient bacterium was Escherichia coli DH5α strain. Escherichia coli DH5α strain containing IFN-β1a gene was cultivated, and pUC57-IFN-β1a was extracted using a plasmid extraction kit.
[0065] 2. Small amount preparation of plasmid DNA:
[0066] (1) Pour the bacterial culture into a 1.5ml microcentrifuge tube, centrifuge at 12,000rpm for 30s in a microcentrifuge, and pour out the culture solution;
[0067] (2) Resuspend and wash the precipit...
Embodiment 2
[0088] Gel recovery and double digestion of the PCR product of embodiment 2
[0089] Make agarose gel with TAE buffer, and perform agarose gel electrophoresis for the target gene. Cut out the agarose gel containing the target gene under ultraviolet light, and absorb the liquid on the surface of the gel with a paper towel. In order to speed up the subsequent melting of the rubber blocks, the crushed rubber blocks were weighed to obtain a total weight of 0.63g, tube A was 0.32g, and tube B was 0.31g, and the rubber block melting liquid DR-I Buffer was added to tubes A and B respectively 960μl and 930μl, mixed evenly and heated at 75°C to melt the gel. Add 1 / 2 volume of DR-II Buffer to the melted solution of the above-mentioned rubber block to prepare a uniformly mixed solution. Place the Spin Column in the kit on the Collection Tube, transfer the uniformly mixed solution to the Spin Column, centrifuge at 12000rpm for 10min, and discard the filtrate. Then add 500μl RinseA, cen...
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