A kind of stable recombinant cardiac troponin and its encoding gene and application
A technology of cardiac troponin and gene, applied in the field of molecular biology, can solve the problems of poor stability, lack of any, and difficult acquisition methods, etc., and achieve the effect of improving thermal stability
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Embodiment 1
[0032] Cloning of cTnI-C complex gene
[0033] According to the cTnI-C complex nucleotide sequence (SEQ ID No.3) published in the patent US6475785B1, it was synthesized by Huada Gene. The gene name is ICH, and the 5' end of the nucleotide sequence has an EcoRI enzyme cutting site point, with 6 histidines, a stop codon and a BamHI restriction site at the 3' end. The amino acid sequence of the encoded protein is shown in SEQ ID No.4.
[0034] The ICH gene fragment and the T vector pMD18T (Bao Bioengineering (Dalian) Co., Ltd.) were ligated according to the following system using T4 ligase from NEB Company:
[0035]
[0036]
[0037] The ligation reaction solution after mixing was placed at 25°C for 30 minutes, and then used at 4°C for later use or directly for transformation reaction.
[0038] Add 10 μL of ligation reaction solution to 100 μL E.coli Top10 competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number CB104), mix gently, ice bath for ...
Embodiment 2
[0040] Construction of recombinant expression construct pBV-ICH
[0041] EcoRI and BamHI (Bao Bioengineering (Dalian) Co., Ltd.) double enzyme digestion reaction was performed on the sequencing construct pMD18T-ICH in Example 1 to recover the ICH target gene fragment:
[0042]
[0043] After mixing, the enzyme digestion reaction solution was placed at 37° C. for overnight incubation, and then purified and recovered by the method in Example 1. The pBV220 expression vector was digested with EcoRI and BamHI in the same manner, and the linear pBV220 expression vector fragment was purified and recovered.
[0044] The purified ICH target gene fragment and the linear pBV220 expression vector fragment were ligated according to the following system:
[0045]
[0046] The ligation reaction solution after mixing was placed at 25°C for 30 minutes, and then used at 4°C for later use or directly for transformation reaction.
[0047] Add 10 μL of ligation reaction solution to 100 μL ...
Embodiment 3
[0049] Random mutation of ICH gene by error-prone PCR
[0050] Prepare the error-prone PCR reaction solution according to the following system:
[0051]
[0052]
[0053] in:
[0054] 10×PCR buffer: 20mmol / L MgCl 2 , 200mmol / L KCl, 50mmol / L Tris-HCl, pH 8.3;
[0055] 50×dNTPs mixture: containing 10mmol / L each of dATP, dGTP, dTTP, and dCTP;
[0056] 10×MgCl 2 Solution: 70mmol / L, dissolved in sterile water;
[0057] MnCl 2 Solution: 1mmol / L, dissolved in sterile water;
[0058] rTaq DNA polymerase: purchased from Treasure Bioengineering (Dalian) Co., Ltd., catalog number DR001.
[0059] The error-prone PCR reaction conditions are: pre-denaturation at 95°C for 5 minutes, and then cycle the reaction 20 times according to the following parameters: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, and finally extension at 72°C for 10 minutes
[0060] After the PCR product was subjected to 1% low melting point agaro...
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