Method for rapidly detecting recombinant protein expression quantity
A technology for recombinant protein and expression, which is applied in the field of genetic engineering, can solve the problems of cumbersome operation, difficulty in screening scale, expansion, etc., and achieve the effect of simple operation, convenient follow-up operation and wide applicability
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Embodiment 1
[0052] Construction of expression vector
[0053] 1.1 Acquisition of GFP gene
[0054] 1.1.1 Design of primers for GFP gene synthesis
[0055] Obtain the coding gene of GFP (SEQ ID NO: 1) by the method of whole gene synthesis, add 6xHis tag to the C-terminus of the target fragment; there are BamHI and NotI restriction sites in the upstream and downstream of the target gene respectively: use DNAworks online The program designed 34 primers with a length of 40bp, and obtained the full-length sequence of the GFP gene by PCR assembly and overlap extension PCR.
[0056] Primer treatment: each primer was dissolved in water and diluted to a concentration of 5 μM.
[0057] The 34 primers were divided into two groups for PCR assembly: primers 1-18 (SEQ ID NO: 2-19) and 17-34 (SEQ ID NO: 18-35). Preparation of primer mixture: Take 10 μL each of the first and last primers, and 5 μL each of the middle primers, mix thoroughly, and use them as substrates for the PCR assembly process.
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Embodiment 2
[0121] Screening of high expressing clones
[0122] 2.1 Plate preparation
[0123] Prepare LB agar medium and make two kinds of plates: solid plates added with 100 μg / mL ampicillin and 100 μg / mL ampicillin and 0.2 mM IPTG respectively. The petri dish adopts a square flat plate of 9cm x 9cm.
[0124] Transform the above final expression vector into Escherichia coli BL21(DE3) competent cells. After activation, spread the culture solution on an LB plate containing 100 μg / mL ampicillin and 0.2 mM IPTG, and cultivate overnight at 37°C to obtain expression Single colony with GFP protein. At the same time, a negative control for transforming BL21(DE3) competent cells with the expression vector without GFP was made.
[0125] The single clones of the negative control and positive colonies were picked and planted on the above two LB plates, and cultured overnight at 37°C for subsequent experiments.
[0126] 2.2 Membrane preparation and protein immobilization
[0127] Nitrocellulose...
Embodiment 3
[0132] Validation of expression clone screening methods
[0133] 3.1 Colony PCR method to verify the insertion of the target fragment
[0134] Pick a single colony and dissolve it in 20 μL of double-distilled water, heat it at 98°C for 10 minutes, take 3 μL of the sample as a PCR template, and use the head-to-tail primers synthesized by the above gene to detect the insertion of the target gene.
[0135] The 20μL PCR reaction system is as follows:
[0136]
[0137] PCR reaction conditions: 95°C for 3min; (94°C for 24sec, 57°C for 24sec, 72°C for 35sec) x 30cycles; 72°C for 2min. Carry out agarose gel electrophoresis after PCR reaction finishes, and the size of the amplified fragment is about 700bp (such as figure 2 ).
[0138] 3.2 western blot detection
[0139] Pick the single clone that has been transferred into the correct expression vector and plant it on the LB plate. After culturing overnight at 37°C, copy the single clone to the nitrocellulose membrane, lyse it a...
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