Gene engineering bacteria for achieving extracellular high expression of procambarus clarkii cytochrome C oxidase
A technology of genetically engineered bacteria and Procambarus clarkii, applied in the field of genetic engineering, can solve the problems of decreased egg quality, shortened parent molt cycle, increased mortality, etc., and achieve the effect of efficient secretion and expression
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Embodiment 1
[0017] Example 1 Construction of recombinant plasmid pMA0911-CCO.
[0018] The sequence of the CytochromeCoxidase gene of Procambarus clarkii is shown in SEQ ID NO.1 (GenBank accession number: AFQ31579), and the primer Cox-1 was designed: (5'-TCAGGA GTC GAC ATGGCAAGATGAGGAAATTTAGG-3′) and Cox-2: (5′-ACTGAG AAGCTT TTATCAAATGAAGATTAAACATG-3'), Utilize PCR to introduce the DNA coding frame 5' and 3' both sides of CytochromeCoxidase gene into SalI and HindIII restriction enzyme sites (underlined) respectively; The gene was inserted into the expression plasmid pMA0911 (wapA) to construct the recombinant plasmid pMA0911-Cox.
Embodiment 2
[0019] Example 2 SDS-PAGE analysis of Procambarus clarkii CytochromeCoxidase protein secreted and expressed by B. subtilis WB600 genetically engineered bacteria.
[0020] Inoculate a single clone of gene bacteria in LB medium with a kanamycin concentration of 50 μg / mL, and culture overnight at 37°C and 100 r / min. Then inoculate 100 μL of the overnight cultured bacterial solution into 100 mL of fermentation medium (containing 50 μg / mL kanamycin and 0.15 mM copper sulfate), culture on a shaking table at 37 ° C and 200 r / min for 24 h, then take out the fermentation broth, 8000 r / min Centrifuge for 10 min to obtain the supernatant, which is the crude enzyme solution. The crude enzyme solution was further separated and purified by ion exchange chromatography. The crude enzyme solution and the purified enzyme solution were subjected to 12% SDS-PAGE protein electrophoresis. The above-mentioned relevant methods all adopt conventional operation steps.
Embodiment 3
[0021] Example 3 Optimization of fermentation medium conditions.
[0022] The concentration of corn starch, peptone and yeast extract in the fermentation medium was optimized, and the optimum temperature of fermentation was studied. When the concentration of corn starch was 20g / L, the PMCA protein production was the highest, reaching 0.672mg / mL; the concentration of peptone When the concentration of yeast extract is 13g / L, the concentration of yeast extract is 11g / L, and the fermentation temperature is 37℃, the CaM protein production can be further increased to about 0.811mg / mL.
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