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Gene recombinant protein for pacific oyster IgSF molecule CgCAICP1 as well as preparation method and application thereof

A technology of gene recombination and protein recombination, applied in the field of molecular biology, to achieve the effect of improving the phagocytosis rate

Active Publication Date: 2018-05-04
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Gene recombinant protein for pacific oyster IgSF molecule CgCAICP1 as well as preparation method and application thereof
  • Gene recombinant protein for pacific oyster IgSF molecule CgCAICP1 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
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 gene recombinant protein for a pacific oyster IgSF molecule CgCAICP1. An amino acid sequence of the gene recombinant protein is shown as SEQ ID NO.1. The preparation method sequentially comprises the following steps: performing PCR amplification on coding region fragments of the pacific oyster IgSF molecule CgCAICP1 by using specific primers P1 and P2, wherein a DNA sequence of the primer P1 is shown as SEQ ID NO.2, and a DNA sequence of the primer P2 is shown as SEQ ID NO.3; performing NcoI and Hind III enzyme digestion on the PCR amplification product and a pET32a vector, connecting the PCR amplification product and the vector by T4 ligase, transforming, and sequencing and identifying the recon; transferring the recon into an Escherichia coli Transetta (DE3) expression strain for performing induction culture; purifying and renaturating, thereby obtaining the gene recombinant protein. The gene recombinant protein for the pacific oyster IgSF molecule CgCAICP1disclosed by the invention can be applied to preparation of medicines 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 Applications(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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