Target thrombolytic protein expressing plasmid and its construction
A technology for expressing plasmids and annexin, applied in the field of medicine and biology, can solve the problems of easy thrombosis, large amount of treatment, lack of specific affinity for fibrin, etc.
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Embodiment 1
[0031] Embodiment 1 The acquisition of fusion gene anx32-scu-PA (144~411)
[0032] See figure 1 , the gene encoding annexin 32 and the gene encoding amino acids 144-411 of single-chain urokinase were respectively amplified by PCR; then the fusion gene anx32-scu-PA(144-411) was obtained by connecting by overlapping region extension . Specific steps are as follows:
[0033] (1) The entire coding gene of annexin 32 gene was amplified with primer a and primer b. Wherein primer a is 5'TG GAATTC ATGGCCTACTGTCGCTCCC3', the primer is consistent with the 5' end of the anx32 coding sequence, and at the same time introduces the EcoRI restriction endonuclease site. Primer b is: 5'GCCACACTGAAATTTTAA / TGCAGGGCCGATGAG3', wherein 18 bp at the 5' end is consistent with the 5' end of the single-stranded urokinase coding sequence, and the latter 15 bp is complementary to the 3' end of the anx32 gene. PCR reactions were performed using high-fidelity pfu DNA polymerase with a dNTP concentrat...
Embodiment 2
[0036] Example 2 Construction of expression plasmid pJM-anx32-scuPA
[0037] The fusion gene anx32-scu-PA (144-411) amplified by the above method was sequenced using primers a and d, and the sequencing reaction was completed by Shanghai Jikang Company. After the sequencing was correct, it was digested with EcoRI and SalI, and connected with the vector pJM which was digested with EcoRI and SalI. Restriction enzymes and T for digestion and ligation reactions 4 All ligases were purchased from TaKaRa Company, and the reaction was carried out using the system recommended by the enzyme instructions. The heat-inducible expression vector pJM utilized in the present invention has a full length of 4.9Kb (kilobase pairs), containing phage P R P L Promoter, heat-inducible gene CI1857, plasmid origin of replication (ori) and ampicillin resistance gene (AP r ).
[0038] The specific reaction process is as follows: EcoRI and SalI were used to perform double enzyme digestion on the above...
Embodiment 3
[0039] The preparation of embodiment 3 engineering bacteria
[0040] The above ligated product was transformed into Escherichia coli strain K802 to obtain engineering strain K802 (pJM-anx32-scuPA).
[0041] The specific steps are as follows: in 50ml of LB medium (containing 1% tryptone, 0.5% yeast extract and 1% sodium chloride, pH7.0), the host bacterium K802 was shaken at 37°C until OD 600 At 0.4, centrifuge at 4000rpm at 4°C to collect the host bacteria; remove the supernatant and resuspend with 1-2ml of ice-cooled 100mmol / L calcium chloride solution to obtain competent bacteria. Cool 100 ng of the plasmid constructed above and competent bacteria in an ice bath for 30 minutes, heat shock at 42° C. for 90 seconds, and then cool in an ice bath for 10 minutes. Add 0.8ml of LB, incubate at 37°C for 1 hour, take 0.2ml of the transformation product and smear it on the LB agar plate containing the corresponding antibiotic, and incubate at 37°C for 12-15 hours to obtain a single-c...
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