Gene engineering bacteria capable of resisting reactive oxygen species (ROS) oxidative stress and efficiently secreting express foreign protein and construction method of gene engineering bacteria
A genetically engineered bacteria, oxidative stress technology, applied in microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of easy formation of insoluble inclusion bodies, expensive industrial production, and inability to express structures, etc. The effect of reducing ROS, avoiding damage, and improving cell viability
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Embodiment 1
[0020] Example 1: Functional identification of mpr1 transacetylation
[0021] 1. According to the gene sequence of mpr1 registered on NCBI (Genbank No. 8198718 ) designed primers to obtain the N-acetyltransferase gene mpr1 from the total DNA of P. pastoris GS115 by PCR, and connected it to the T vector. The ligated product was transformed into Escherichia coli JM109, and the transformed product was coated on an LB plate containing 100 mg / L ampicillin. After culturing overnight at 37°C, colonies were selected and inserted into LB liquid medium. After 8-10 hours, the plasmid was extracted, and the plasmid was sequenced and identified. The results show that the gene has a full length of 627 nucleotides, which is exactly the same as the gene sequence of mpr1 registered on NCBI, and the size of the electrophoresis band is as follows: figure 1 .
[0022] 2. The mpr1 gene was obtained by PCR from the T carrier, and the plasmid pQE30 was digested and ligated to construct the recombi...
Embodiment 2
[0023] Embodiment 2: Construction of mpr1 overexpression genetically engineered bacteria
[0024] 1. The pPICZA plasmid and the recombinant T vector were digested with BamHI and EcoRI respectively. After the digested products were recovered by tapping the gel, they were ligated with T4 ligase to obtain pPICZA-mpr1. The size of the plasmid fragment was verified by enzyme digestion.
[0025]2. The recombinant expression vector pPICZA-mpr1 was linearized by Sac I single enzyme digestion and then electrotransformed into P. pastoris KM71, and the recombinant bacteria were coated with a YPD plate containing 2 mg / mL Zeocin resistance. Pick a single colony on the resistant plate and inoculate it in 10 mL of YPD medium containing Zeocin antibiotic (peptone 20g / L, yeast extract 10g / L, glucose 20g / L), culture at 30°C overnight, and extract the yeast genome for PCR identification of strain recombination Correct, name it P.pastoris / pPICZA-mpr1, and store it in a glycerol tube. The overexp...
Embodiment 3
[0026] Example 3: This example shows that the overexpression of mpr1 can significantly improve the ability of cells to resist ROS oxidative stress and help to increase the expression level of foreign proteins.
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