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A kind of pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody and application thereof

A monoclonal antibody and polymerase technology, applied in the field of bioengineering, to achieve the effects of convenient production, good sealing effect and simple production

Active Publication Date: 2020-09-29
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no Pfu 3'-5'exonuclease activity blocking monoclonal antibody on the market, and there is no hot start blocking antibody against Pfu mutant polymerase

Method used

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  • A kind of pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody and application thereof
  • A kind of pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody and application thereof
  • A kind of pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Induced expression and affinity purification of recombinant protein

[0052] (1) The recombinant expression vector pET-28a-Canace was transformed into a Rosetta E. coli strain to induce the expression of Canace protein.

[0053] Pick a monoclonal Rosetta colony in 5 mL of kanamycin-resistant LB medium, culture overnight, expand to 1 L of kanamycin-resistant LB, and culture to OD 600 0.8 to 1.0, add 400 μL of 1 M IPTG, and induce for 4 hours at 37°C. Put the bacterial solution into a 1 L centrifuge cup, 4000r / min, 15 min. Discard the supernatant, add 20 mL PBS to resuspend the cells, and freeze in a -80°C refrigerator.

[0054] (2) The Canace protein was purified by nickel column affinity chromatography, and the protein purification effect was identified by SDS-PAGE.

[0055] Thaw the frozen bacterial solution in a water bath at room temperature, and ultrasonically break at 300 W, 5s / 5s, for 1 h. Transfer the crushed bacterial solution to a high-speed centr...

Embodiment 2

[0056] Example 2 Mouse immunization and serum titer determination

[0057] Using purified Canace protein as the immunogen, 6-week-old Balb / c female mice were immunized according to the immunization procedure, and the titer of the immunized mice was detected by indirect enzyme-linked immunosorbent assay (ELISA). The specific method is:

[0058] Three six-week-old Balb / c mice were immunized, Canace protein was emulsified with Freund's complete adjuvant for the first immunization, and Canace protein was emulsified with Freund's incomplete adjuvant for subsequent immunizations, and the immune dose was 100 μg per mouse Protein, immunized once a week, immunized for two months, by subcutaneous multi-point injection. Orbital blood was collected before each immunization after the third immunization, and the serum titer was detected by enzyme-linked immunosorbent assay (ELISA). The more specific method is to coat the plate with 1 μg / mL Canace protein (dissolved in PBS), block with 5% ...

Embodiment 3

[0059] Example 3 Cell Fusion and Hybridoma Screening

[0060] Splenocytes from immunized mice were fused with SP2 / 0 cells, and positive hybridoma cell lines were screened by ELISA. The specific method is:

[0061] Cultivate a sufficient amount of SP2 / 0 in advance, not less than 10 8 to ensure that the cells are in the logarithmic growth phase. Collect SP2 / 0 and wash three times with serum-free RPMI1640 medium. The spleens of the mice immunized with shock were taken, and the splenocytes were obtained by grinding, and the splenocytes were washed three times with RPMI1640 medium. Splenocytes and SP2 / 0 cells were mixed at a ratio of 2:1 to 5:1. 1000r / min, 10 min, remove the medium, add 1 mL PEG1450 into the cells within 1 min, and stir gently while adding. Within two minutes after adding PEG1450, add 2 mL of RPMI1640 culture medium at a uniform speed to terminate the effect of PEG1450, and then add RPMI1640 medium to a volume of 50 mL within 2 minutes. 1000r / min, 10 min, cen...

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Abstract

The invention discloses a Pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody, which comprises a light chain variable region containing LCDR1, LCDR2 and LCDR3 sequences and a HCDR1, HCDR2 and HCDR3 sequence Heavy chain variable region, the sequence of the LCDR1 is shown in SEQ ID NO: 3, the sequence of the LCDR2 is shown in SEQ ID NO: 4, the sequence of the LCDR3 is shown in SEQ ID NO: 5, the The sequence of HCDR1 is shown in SEQ ID NO: 7, the sequence of HCDR2 is shown in SEQ ID NO: 8, and the sequence of HCDR3 is shown in SEQ ID NO: 9. The invention also discloses the application of the monoclonal antibody. The blocking effect of the monoclonal antibody of the invention is good, and the blocking rate reaches nearly 100%.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular, it relates to a Pfu mutant polymerase 3'-5' exonuclease activity blocking monoclonal antibody and its application. Background technique [0002] Polymerase Chain Reaction (PCR) is a method for synthesizing double-stranded DNA in vitro. Its principle is similar to the replication process of natural DNA, and its specificity mainly depends on the specific primers complementary to both ends of the target fragment. A typical PCR reaction has three steps: denaturation, annealing, extension, and multiple cycles of reaction to obtain the target fragment. [0003] At present, there are many methods to improve the specificity of PCR amplification, and the simplest method is to use hot-start enzyme amplification. The optimum temperature for ordinary DNA polymerase is 72°C, at this time the enzyme activity is the best, below this temperature, the enzyme has weak activity, 5'-3' polymer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C07K1/22C12N9/12C12Q1/6848
CPCC07K16/40C07K2317/565C12N9/1252C12Q1/6848C12Y207/07007C12Q2521/101C12Q2531/113
Inventor 易红飞周其好腾以刚王亚茹沈晓玲袁灿灿宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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