A gene encoding ifnα protein, recombinant vector pelsh-ifnα, recombinant Lactobacillus casei and application thereof
A technology of recombinant cheese and recombinant vector, applied in the direction of lactobacillus, recombinant DNA technology, application, etc., can solve the problems of protection effect and effectiveness difference, and achieve the effect of extending the shelf life
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[0030] In the present invention, the preparation method of the recombinant vector pELSH-IFNα preferably includes the following steps: double enzyme digestion of the gene encoding the IFNα protein and the plasmid pELSH, recovery of the digestion products, and enzyme ligation of the digestion products to obtain the recombinant vector pELSH-IFNα.
[0031] In the present invention, the gene encoding IFNα protein and the plasmid pELSH double enzyme digestion system are shown in Table 1; the temperature of the enzyme digestion is preferably 35-40°C, more preferably 37°C; the time of the enzyme digestion is preferably 1.5 ~3h, more preferably 2h.
[0032] Table 1 Gene encoding IFNα protein and plasmid pELSH double enzyme digestion system
[0033]
[0034] In the present invention, the kit for recovering enzyme-cut products is preferably a PCR cleaning and recovery kit, which is purchased from OMEGA Company; after the recovery of enzyme-cut products, it preferably also includes rem...
Embodiment 1
[0044] Example 1 Construction of recombinant vector pELSH-IFNα
[0045] 1. Double digestion of target fragment and plasmid pELSH
[0046] For the double enzyme digestion system, see Table 1 of the above scheme. Digestion at 37°C for 2 hours. Use the PCR cleaning and recovery kit to recover the digested product, remove the enzyme and other substances in the reaction system, and wait for the agarose gel electrophoresis to verify the success.
[0047] 2. Enzyme Ligation of Target Fragment and Vector Fragment
[0048] For the enzyme-linked system, see Table 2 of the above scheme. Enzyme chain reaction was carried out at 16°C for 1 hour to obtain the recombinant vector pELSH-IFNα.
Embodiment 2
[0049] Embodiment 2 constructs recombinant Lactobacillus casei
[0050] The recombinant vector pELSH-IFNα obtained in Example 1 was electrotransformed into Lactobacillus casei competent cells, and after standing at 37°C for resuscitation, the MRS plate containing erythromycin was coated, and then cultured upside down at 37°C for 2 days, then screened, verified, and positive Recombinant. For verification results, see figure 2 and image 3 ,in figure 2 It is the PCR verification of JM109-pELSH-hIFNα recombinant bacterial liquid, M is Trans2KPlus DNA Marker, and 1-10 are 10 transformants picked from the plate. The empty load size of pELSH is 640bp, and the PCR product size of pELSH-hIFNα recombinant plasmid is 1105bp. From the results of agarose gel electrophoresis, it can be seen that the band is single and bright, and the PCR product fragments in the 10 lanes are of the same size, which is in line with expectations. image 3 Shows the double enzyme digestion verification...
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