Ribonuclease and its preparation method, application and efficacy detection method, recombinant vector and recombinant engineering bacteria and its construction method
A technology of ribonuclease and recombinant vector, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problem of poor tumor lethality
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[0117] The preparation method of ribonuclease according to one embodiment includes the following steps: amplifying and culturing the above-mentioned recombinant engineering bacteria, adding an inducer to continue culturing, separating solid and liquid after the cultivation, collecting the supernatant, and obtaining ribonuclease.
[0118] In one of the embodiments, the step of amplifying and cultivating the above-mentioned recombinant engineered bacteria includes: cultivating the recombinant engineered bacteria with a medium containing glycerol, adding a certain amount of glycerol at a constant rate to continue the culture after the glycerol is exhausted, and when the glycerol is exhausted again, Then starve and culture for 0.5h~1h. Further, after the cultivation, the wet weight of the bacteria reaches at least 130g / L-200g / L.
[0119] In a specific example, the inducer is methanol. Further, the method of adding the inducer to continue the cultivation is to adjust the flow spee...
Embodiment 1
[0144] Construction of recombinant vector
[0145] (1) Restriction endonuclease SacI and restriction endonuclease NotI were used to double-digest pPIC9K vector to obtain linearized pPIC9K vector. Among them, the schematic diagram of the structure of the pPIC9K vector is as follows: figure 2 shown. figure 2 It is a schematic diagram of the structure of the pPIC9K vector.
[0146] (2) Use the amplification primers in Table 1 to perform PCR amplification on the signal peptide sequence and ribonuclease respectively, detect the position of the DNA band by agarose gel electrophoresis, recover the target fragment from the gel, and obtain the first PCR amplification respectively fragment and the second PCR amplified fragment. Wherein, the signal peptide sequence is shown in SEQ ID No.8. The ribonuclease is one of the ribonucleases whose amino acid sequences are shown in SEQ ID No.1 to SEQ ID No.7. The first PCR amplified fragment is the amplified product of the signal peptide s...
Embodiment 2
[0156] Construction of Pichia pastoris expression vector for ribonuclease
[0157] (1) Digest the expression vector of the ribonuclease of Example 1 with restriction endonuclease SacI to linearize the expression vector, and recover the linearized expression vector by ethanol precipitation. 4 μg of the linearized expression vector was mixed with 80 μL of competent Pichia pastoris GS115 (purchased from Invitrogen), placed in a 0.2 cm electroporation cuvette, and incubated on ice for 5 min. Under the conditions of voltage 1.5KV, capacitance 25mF, resistance 200Ω. Immediately after electroporation, 1 mL of pre-cooled 1M sorbitol aqueous solution was added and mixed evenly. Spread on MD culture plate and incubate at 30°C until clones grow out. Take an appropriate amount of sterile water and add it to the MD culture plate, and scrape off the clones on the MD culture plate with a sterile coating stick. Cell density was measured with a spectrophotometer.
[0158] (2) Prepare YPD p...
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