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Long oyster igsf molecule cgcaicp1 gene recombinant protein, preparation method and application

A technology of gene recombination and recombination protein, applied in the field of molecular biology, can solve the problem of no preparation method etc.

Active Publication Date: 2021-05-18
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, so far, there are no relevant reports about the recombinant protein of the long oyster IgSF molecule CgCAICP1 gene, its preparation method and its application in the preparation of drugs for inhibiting Gram-negative bacteria

Method used

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  • Long oyster igsf molecule cgcaicp1 gene recombinant protein, preparation method and application
  • Long oyster igsf molecule cgcaicp1 gene recombinant protein, preparation method and application

Examples

Experimental program
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Effect test

experiment example 1

[0036] Experimental example 1: Long oyster recombinant protein r C g CAICP1 Bacterial Binding Activity Detection

[0037] Based on the western blotting method, the recombinant protein was tested against two Gram-negative bacteria (Vibrio resplendent and Escherichia coli), two Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and a fungus Pichia pastoris binding activity. The sources of the strains used are as follows: Vibrio splendidus ( Vibrio splendidus JZ6 ) was purchased from Beijing Microorganism Culture Collection Center, Escherichia coli ( Escherichia coli ) was purchased from Beijing Quanshijin Company, Staphylococcus aureus ( Staphylococcus aureus ) was purchased from Beijing Microorganism Culture Collection Center, Micrococcus luteus ( Micrococcus luteus ) was purchased from Beijing Microbial Culture Collection Center, Pichia pastoris ( Pichia pastoris GS115 ) were purchased from Invitrogen.

[0038] The specific operation is as foll...

experiment example 2

[0057] Experimental example 2: Long oyster recombinant protein r C g Detection of CAICP1 pro-phagocytosis activity

[0058] Three kinds of microorganisms (Vibrio splendidus, Micrococcus luteus, and Pichia pastoris) were labeled with fluorescein isothiocyanate (FITC), and then the phagocytosis efficiency of oyster blood lymphocytes was detected by counting with a fluorescence microscope.

[0059] The strain source is as above.

[0060] The specific operation is as follows:

[0061] (1) Cultivate and collect the above three microorganisms separately;

[0062] (2) Use formaldehyde to mix with microorganisms and fix for 10 min;

[0063] (3) Collect the bacteria by centrifugation at 4000×g for 10 minutes. 0.1 M NaHCO 3 After washing three times, incubate in 0.1M NaHCO containing 0.1 mg / ml FITC 3 Incubate at 25 °C for 2 h with gentle shaking;

[0064] (4) After centrifuging and discarding the supernatant, use TBS buffer to wash the microorganisms until they are colorless;

...

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Abstract

The invention discloses a long oyster IgSF molecule CgCAICP1 Gene recombinant protein, the amino acid sequence is shown in SEQ ID NO.1. The preparation method is carried out according to the following steps in sequence: Use primers P1 and P2 to pair the long oyster IgSF molecule CgCAICP1 The coding region fragment was amplified by PCR; the PCR amplified product was combined with pET32a vector via Nco I and Hind After digestion with enzyme III, ligate with T4 ligase, transform, sequence and identify recombinants; transfer the above recombinants into Escherichia coli Transetta (DE3) expression strains for induction and culture, then purify and renature. The long oyster IgSF molecule CgCAICP1 The gene recombinant protein can be applied to the preparation of drugs for inhibiting Gram-negative bacteria.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a long oyster IgSF molecule CgCAICP1 Gene recombinant protein, preparation method and application. Background technique [0002] Immunoglobulin superfamily IgSF molecules, mainly produced by B lymphocytes, are an important component of vertebrate adaptive immunity. They can produce a variety of antigen-binding epitopes through a series of molecular mechanisms and bind to corresponding Antigen determinant specific binding. Although invertebrates do not possess the antibody-based adaptive immune system found in vertebrates, they are still able to form an effective immune response to pathogenic invasion. Studies have found that the main ways of IgSF molecules to perform functions include the following four types: a. pattern recognition; b. microbial agglutination; c. phagocytosis opsonization; d. cell agglutination and anti-agglutination. For example, hemolin in insects...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C12N15/70C12N15/66A61K38/17A61P31/04
CPCA61K38/00C07K14/70503C12N15/66C12N15/70
Inventor 宋林生刘冬杨衣启麟宋小瑞王玲玲
Owner DALIAN OCEAN UNIV
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