Transformed transferrin DNA binding domain, recombinant DNA polymerase and preparation method
A technology of transferrin and lactotransferrin, which is applied in the field of recombinant DNA polymerase and the preparation and transformation of the DNA binding domain of transferrin to achieve the effects of reducing costs, increasing benefits, and facilitating operation
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Embodiment 1
[0027] In this example, the DNA binding domains of 3 kinds of human-derived, 3 kinds of mouse-derived, and 1 kind of chicken-derived transferrin were detected, and the amino acid sequences were as follows:
[0028] Human LTF (HLTF): GRRRRSVQWCAVSQPEATKCFQWQRNMRKVR (SEQ ID No. 1)
[0029] Human TF (HTF): AVPDKTVRWCAVSEHEATKCQSFRDHMKSVI (SEQ ID No. 3)
[0030] Human MTF (HMTF): VLGGMEVRWCATSDPEQHKCGNMSEAFREAG (SEQ ID No. 5)
[0031] Murine LTF (MLTF): LAKATTVQWCAVSNSEEEKCLRWQNEMRKVG (SEQ ID No. 2)
[0032] Murine TF (MTF): AVPDKTVKWCAVSEHENTKCISFRDHMKTVL (SEQ ID No. 4)
[0033] Murine MTF (MMTF): VVCVMEVQWCTISDAEQQKCKDMSEAFQGAG (SEQ ID No. 6)
[0034] Chicken OTF (GOTF): APPKSVIRWCTISSPEEKKCNNLRDLTQQER (SEQ ID No. 7)
[0035] The transformation method of the above-mentioned DNA binding domain is as follows:
[0036] Since there are 2 C amino acids in the DNA binding domain of transferrin that can form disulfide bonds, the disulfide bonds are unstable at high temperature, so...
Embodiment 2
[0046] The transferrin DNA-binding domain obtained in Example 1 can be further modified, and the amino acids at positions 1-5 of the DNA-binding domain of transferrin can be changed to KFKYK, and positions 28-31 are changed to KKVK, wherein 1-5 The amino acid modification of the position is relatively important. The performance of DNA polymerase can be further improved.
[0047] The amino acid sequence of the DNA-binding domain of transferrin after the second step transformation is as follows:
[0048] Human LTF" (HLTF"): KFKYKSVQWRAVSQPEATKAFQWQRNMKKVK (SEQ ID No. 15)
[0049] Human TF" (HTF"): KFKYKTVRWRAVSEHEATKAQSFRDHMKKVK (SEQ ID No. 16)
[0050] Human MTF" (HMTF"): KFKYKEVRWRATSDPEQHKAGNMSEAFKKVK (SEQ ID No. 17)
[0051] Murine LTF" (MLTF"): KFKYKTVQWRAVSNSEEEKALRWQNEMKKVK (SEQ ID No. 18)
[0052] Murine TF" (MTF"): KFKYKTVKWRAVSEHENTKAISFRDHMKKVK (SEQ ID No. 19)
[0053] Murine MTF" (MMTF"): KFKYKEVQWRTISDAEQQKAKDMSEAFKKVK (SEQ ID No. 20)
[0054] Chicken OTF" (GO...
Embodiment 3
[0056] The transformed DNA-binding domains of transferrin in Examples 1 and 2 were coupled with Taq DNA polymerase to form a recombinant heat-resistant DNA polymerase.
[0057] (1) Amplification efficiency comparison experiment:
[0058] Purified Taq, HLTF'-Taq, HLTF'-Taq, HTF'-Taq, HTF'-Taq, MMTF'-Taq, MMTF'-Taq, GOTF'-Taq, GOTF'-Taq serially diluted in half, take each A 1.5 kb DNA fragment was amplified in a 20 μL reaction volume at a dilution of 1 μL (25 cycles). like figure 1 As shown, lanes 1-6 of agarose gel electrophoresis (loading 5 μl / lane) were serially half-diluted (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32) amplification results.
[0059] (2) Amplification speed comparison experiment: such as figure 2 As shown, Taq, HLTF'-Taq, HLTF'-Taq, HTF'-Taq, HTF'-Taq, MMTF'-Taq, MMTF'-Taq, GOTF'-Taq, GOTF'-Taq under 20 s extension conditions 1.5kb, 2.5kb, 3.5kb and 4.5kb DNA fragments were amplified.
[0060] The experimental results show that the recombinant heat-resistant DNA po...
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