Preparing method of xanthine oxidase for clinical detection
A technology of xanthine oxidase and xanthine dehydrogenase, which is applied in the field of molecular biology, can solve the problems of high production cost, large application restrictions, and single source, and achieve low production cost, easy scale-up, and high-efficiency expression Effect
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[0021] A kind of preparation method of xanthine oxidase for clinical detection of the present invention, the method comprises the following steps:
[0022] Step 1. Three genes xdhA, xdhB and xdhC are introduced into E. coli, and the three genes are co-expressed in E. coli to obtain xanthine dehydrogenase; wherein: xdhA (taken from Gene ID: 947116), xdhB (taken from Gene ID: 947116), xdhB (taken from Gene ID: 947205) and xdhC (from Gene ID: 945148) are E. coli genes.
[0023] The specific process is as follows: connect the xdhA and xdhC genes to the E. coli co-expression vector pETDuet-1 to construct the recombinant plasmid pETDuet-1-xdhA-xdhC, and connect the xdhB gene to the E. coli expression vector pACYCDuet-1 to construct the recombinant plasmid pACYCDuet- 1-xdhB; then the above two recombinant plasmids were co-transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli expressing xanthine dehydrogenase.
[0024] A histidine tag is only added to the...
Embodiment 1
[0035] Cloning of embodiment 1 xanthine dehydrogenase gene:
[0036] Escherichia coli K12 was cultured overnight at 37°C in LB medium, the Escherichia coli cells were collected by centrifugation, and the genomic DNA of Escherichia coli was extracted using a bacterial genome extraction kit. The gene sequences of the three subunits xdhA, xdhB and xdhC of xanthine dehydrogenase were obtained by PCR amplification using the extracted genomic DNA as a template. Primers were designed according to the gene sequences of the three subunits xdhA (from Gene ID: 947116), xdhB (from Gene ID: 947205) and xdhC (from Gene ID: 945148) of Escherichia coli xanthine dehydrogenase in the NCBI GenBank database .
[0037] The primer sequence of PCR amplification xdhA gene is:
[0038] Upstream primer xdhA-F: '-AAGTggatccAATGCGCGTCGTCGATGCCATTGC-3';
[0039] Downstream primer xdhA-R: '-TACTgcggcccgcTTAAATCAATCCTGCCAGATG-3';
[0040] The PCR amplification conditions were: 94°C for 4min; 94°C for 1m...
Embodiment 2
[0051] Construction of recombinant expression plasmids pETDuet-1-xdhA-xdhC and pACYCDuet-1-xdhB;
[0052] In order to co-express the three subunits xdhA, xdhB and xdhC of xanthine dehydrogenase in Escherichia coli, we connected the xdhA and xdhC genes with the co-expression vector pETDuet-1 to construct the vector pETDuet-1-xdhA-xdhC, Such as figure 2 As shown in A. At the same time, the xdhB gene and the expression vector pACYCDuet-1 were connected to construct the vector pACYCDuet-1-xdhB, such as figure 2 Shown in B. The specific experimental process is as follows:
[0053] Such as figure 2 As shown, using Escherichia coli genomic DNA as a template, the xdhA gene was amplified by PCR, and then the pure xdhA gene was obtained through the DNA gel recovery kit. Plasmid pETDuet-1 and xdhA genes were digested with BamHI and NotI at 37°C for 4h, respectively, and then recovered by DNA gel recovery kit. Ligate the recovered plasmid and xdhA gene overnight at 16°C, transfer...
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