Pgk1 protein, recombinant plasmid for expressing Pgk1 protein, recombinant probiotic for expressing Pgk1 protein and application
A technology of recombinant plasmids and probiotics, applied in the direction of peptide/protein components, applications, recombinant DNA technology, etc., to improve neurological behavior, significant therapeutic effect, and reduce the volume of cerebral infarction
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[0059] Example 1 Preparation of recombinant plasmids expressing Pgk1 and recombinant probiotics expressing Pgk1
[0060] 1. Primer Synthesis
[0061] According to the nucleotide sequence of the gene Pgk1 (shown in SEQ ID NO. 2), and compared with the pET-28a plasmid map, design specific primers, and insert restriction endonuclease sites BamHI and EcoRI into the primers, the specific information See Table-1. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.; the primers were dissolved in sterile deionized water to prepare a concentration of 10 μmol / L.
[0062] Table 1 Primer information and synthesis
[0063]
[0064] 2. Construction of Recombinant Plasmids
[0065] The pET-28a-Pgk1 plasmid was synthesized by Jinweizhi Biotechnology Co., Ltd.
[0066] 3. Transform Escherichia coli DH5α with the recombinant plasmid
[0067] Take out the DH5α competent cells from the -80°C refrigerator, and place them on the ice box for 10-20 minutes to thaw them; ta...
Example Embodiment
[0079] Example 2 Therapeutic effect of recombinant probiotics expressing Pgk1 on ischemic stroke rats
[0080] 1. Preparation and administration time of MCAO model:
[0081] SPF healthy male SD rats, weighing 180-220g, were randomly divided into 7 groups after one week of adaptive feeding. The groups and doses were as follows:
[0082] Sham operation group (SHAM, orally administered an equal volume of 11.1 mg / ml β-lactose solution);
[0083] MCAO ischemia-reperfusion model group (IR, orally administered an equal volume of 0.9% normal saline);
[0084] MCAO model + lactose group (IR + lactose, orally administered an equal volume of 11.1 mg / ml β-lactose solution);
[0085] MCAO model + positive drug group (NBP, oral administration of butylphthalide 20mg / kg / day);
[0086] MCAO model + E.coli Nissle 1917 group: 2.5 mL of Escherichia coli Nissle 1917 was administered by gavage;
[0087] MCAO model + pET-28a-EGFP group: 2.5 mL of recombinant Escherichia coli Nissle 1917 probioti...
Example Embodiment
[0102] Example 3 Therapeutic effect of recombinant probiotics expressing Pgk1 on diabetic mice
[0103] 1. Establishment of C57BL / 6N diabetic mouse model
[0104] SPF grade healthy male C57BL / 6N mice, weighing 18-22g.
[0105] Sixty C57BL / 6N mice were randomly divided into three groups:
[0106] Ordinary diet group: SD group, 8 C57BL / 6N mice. After being fed with common feed for three weeks, intraperitoneal injection of citrate buffer solution (the injection dose is the same as the following STZ solution), fasting and drinking water for 12 hours before the injection of citrate buffer solution, the injection was carried out according to the same time period 9:00am-10:00am, Once a day, continuous injection for five days, 2 hours after the injection of citrate buffer solution and then feeding. Then, the mice were fed with ordinary feed for three weeks and then fasted for 10 hours, and the fasting blood glucose of the mice was detected between 7:30pm and 9:00pm.
[0107]Type 2...
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