Molecular chaperone TSPcpn47 and polynucleotide encoding same
A molecular chaperone, polynucleotide technology, applied in the biological field, to achieve the effect of reducing protein denaturation
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Embodiment 1
[0020] Example 1: Amplification of the molecular chaperone TSPcpn47 gene (Take TSP4 genomic DNA of Thermus bacteriophage as model
[0021] plate)
[0022] (1) The primer sequences used for the amplification of TSP4 phage molecular chaperone TSPcpn47 gene are as follows:
[0023] Forward primer: 5’-CG GAATTC ATGGCACTCTTATCTAAC -3'
[0024] Reverse primer: 5'- ACAT CTCGAG CCATGAGGCCACCTCCT -3'
[0025] (2) The amplification system is as follows:
[0026] Table 1: Amplification reaction system components
[0027]
[0028] (3) The amplification conditions are as follows:
[0029] Mix the reaction system evenly, pre-denature at 95°C for 4 minutes, then denature at 95°C for 30 s, anneal at 60°C for 45 s, and extend at 72°C for 90 s. After 30 cycles, extend at 72°C for 10 min. After the reaction, take 2 μl of the product , carried out electrophoresis analysis in 10g / L agarose gel (see figure 1 ).
Embodiment 2
[0030] Embodiment 2: Gel recovery and purification of PCR product
[0031] ①Create 1.0% agarose gel in the electrophoresis apparatus;
[0032] ② Apply electrophoresis to the PCR product to be separated and purified, and stop the electrophoresis at an appropriate position;
[0033] ③Cut off the gel containing the target fragment under ultraviolet light, and transfer it to a 1.5ml Ep tube;
[0034] ④Recover the target fragment with the Gel Recovery Kit of Soleil Biotechnology Co., Ltd., and the recovery method is operated according to the instructions.
[0035] Submit the gene sequence to the NCBI database Conserved Domain Database protein conservative sequence analysis software for analysis, dCTP deaminase analysis results see figure 2 .
Embodiment 3
[0036] Embodiment 3: Construction of recombinant expression vector
[0037] (1) Preparation of the linear vector pET-28a(+) with cohesive ends and the target fragment
[0038] In order to connect the target gene fragment to the expression vector pET-28a(+), it is necessary to make the target fragment have a fragment with sticky ends, that is, a restriction site. Similarly, in order to enable the target fragment to be inserted into the vector, it is also necessary Make vectors with cohesive ends and make their restriction sites the same.
[0039] A. pET-28a(+) plasmid extraction: use the plasmid extraction kit (Soleibao), the operation steps are as follows:
[0040] Strain activation: Dip the Escherichia coli strain containing the pET-28a(+) plasmid frozen at -80°C with a sterile inoculation loop, inoculate it on the ampicillin LB plate with the third-line method, and incubate at 37°C for 12-16 hours;
[0041] ② Enrichment and collection of bacteria: Add 5 μl of ampicilli...
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