Patatin-like phospholipase as well as coded gene and use thereof
A technology of phospholipase and application, which is applied in the field of genetic engineering, can solve the problems of few reports, and achieve the effect of high catalytic efficiency
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Embodiment 1
[0040] Embodiment 1, polymerase chain reaction (PCR) amplifies the purification of plp gene and amplified product
[0041] Primer 1 and primer 2 containing NdeI and NotI restriction sites were designed, and the subcloned DNA with ester degradation activity was used as a template to clone the full-length gene sequence of patatin-like phospholipase. The sequence of primer 1 is shown in SEQ ID NO.3 The sequence of primer 2 is shown in SEQ ID NO.4; the PCR reaction was carried out in a 50 μL system, and the reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, extension at 72°C for 1 minute, and 30 cycles Afterwards, a final extension at 72°C for 10 min. The amplified product was detected by 0.8% agarose gel electrophoresis, and an obvious positive amplification band was found, that is, a DNA fragment with a size similar to the theoretical size. Cut off the gel block containing the target gene and p...
Embodiment 2
[0042] Embodiment 2, the construction of expression vector
[0043] The recovered plp fragment and the vector pET-28a(+) were subjected to double enzyme digestion (NdeI and NotI) respectively, and then the rubber was tapped for recovery, and the recovered plp fragment was ligated and transformed into Escherichia coli DH5α competent cells. Cultivate overnight on the LB plate containing kanamycin resistance, pick a single colony and insert it into the LB liquid medium containing kanamycin resistance, extract the plasmid after culturing for 10-14 hours, and perform enzyme digestion to verify the insertion of the target fragment The plasmid was sequenced, and the correctly sequenced plasmid was named pET-28a(+)-plp. Transform the recombinant plasmid pET-28a(+)-plp into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21(DE3) / pET-28a(+)-plp containing the recombinant plasmid pET-28a(+)-plp .
Embodiment 3
[0044] Example 3, Induced Expression of Recombinant Plasmids in Escherichia coli
[0045] Pick a single DH5α Escherichia coli containing the recombinant plasmid and inoculate them in 3mL LB liquid medium (containing Kan30μg / L), shake and culture at 37°C overnight, extract the plasmid the next day, and transform the recombinant plasmid into the expression host strain BL21 (DE3) For the competent cells, 100 μL of the bacterial solution was evenly spread on the surface of the LB plate containing Kan (final concentration: 30 μg / L) with a glass rod, and cultured upside down at 37°C overnight. Then, when the bacteria grow out, randomly pick a single colony containing the recombinant plasmid, inoculate it in 3 mL of Kan-containing LB culture medium for overnight culture at 37°C, and transfer it to 3 mL of Kan-containing LB culture medium on the next day according to the inoculum size of 1%. medium, cultured at 37°C to OD 600 For 1.2, no inducer was added to a culture tube containing...
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