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Branchiostoma belcheri C-type lectin BjCTL4 gene and application thereof

A sequence and selection technology, applied in the fields of application, gene therapy, genetic engineering, etc., can solve the problem of lack of adaptive immune system in amphioxus

Active Publication Date: 2021-09-10
GUANGDONG FOOD & DRUG VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Amphioxus lacks the adaptive immune system similar to vertebrates, but it can still survive healthy in the marine environment full of pathogenic microorganisms, mainly relying on its effective and specific immune system

Method used

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  • Branchiostoma belcheri C-type lectin BjCTL4 gene and application thereof
  • Branchiostoma belcheri C-type lectin BjCTL4 gene and application thereof
  • Branchiostoma belcheri C-type lectin BjCTL4 gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] General method and steps of embodiment 1

[0048] cDNA analysis method

[0049] Alignment analysis was completed using ClustalX (1.83) software and Gene Doc software, and phylogenetics analysis (phylogenetics) was basically completed using MEGA 4.1 (Beta) software.

[0050] gene cloning

[0051] The whole fish of Qingdao amphioxus was taken, and the total RNA was extracted according to the instructions of Invitrogen Company TRIzol Reagent Kit. Take 1 μg of total RNA, and perform reverse transcription to synthesize the first strand according to the instructions of TOYOBO's First Strand cDNA Synthesis Kit ReverTraAce-α-TM. According to Invitrogen's GeneRacerTM Kit, perform RNA dephosphorylation RACE, decapping reaction, RNA oligo ligation and mRNA reverse transcription to synthesize a cDNA strand, and use Takara's LA Taq polymerase to perform PCR amplification according to the reaction system of GeneRacerTM Kit. ORF box genes were amplified using Takara's LATaq polymer...

Embodiment 2

[0097] Example 2 cDNA full-length amplification and sequence analysis

[0098] A molecule containing CTLD was found in the digestive tract cDNA library of Qingdao amphioxus. Sequence analysis showed that the 5' and 3' ends of the gene fragment were incomplete. The full-length sequence of the gene was obtained by RACE amplification and named BjCTL4.

[0099] The sequence of BjCTL4 is shown in SEQ ID NO.:1.

[0100]

[0101]

[0102]

[0103] Signal sequences are shown as shaded areas. CTLD is underlined. "WIGL" and the mannose / glucose recognition site (QPD / EPN) are boxed.

[0104] Alignment analysis of the BjCTL4 protein molecule and the CTLD of representative human CTLs revealed that this CTL-like molecule of amphioxus has the basic characteristics of traditional CTLs, including two or three pairs of conserved disulfide bonds, and the characteristic motif WIGL Similar motifs of and with Ca 2+ Related sugar-binding motifs QPD / EPN and similar motifs of WND ( figure...

Embodiment 3

[0105] Example 3 CTLDs sequence analysis of BjCTL4

[0106] The full length of the BjCTL4 gene is 2,059bp, encoding 475 aa. SMART prediction results show that the protein encoded by this gene is composed of N-terminal signal peptide, several epidermal growth factor EGF domains and a CTLD, which is a secreted lectin molecule ( figure 2 ).

[0107] Sequence analysis of the protein encoded by BjCTL4 revealed that the molecule contains three pairs of cysteines, which belong to secreted long-circle CTLs. Their original and simple structure already has most of the characteristics of traditional CTL molecules, including conserved cysteines and WIGL motif, and characteristic motifs EPN / QPD and WND motif (BjCTL4 is EPT). The motifs of EPN and WND are related to calcium ion-dependent sugar binding, and the EPN motif of some CTL molecules may be replaced by the conserved motif of QPD. The proline in the middle of the two motifs of EPN / QPD is highly conserved, which separates the two ...

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Abstract

The invention belongs to the field of biology, and provides a Branchiostoma belcheri C-type lectin BjCTL4 gene and application thereof. A protein coded by the C-type lectin BjCTL4 gene is found for the first time, a CTL-like structural domain of the protein has basic characteristics of traditional CTL, the protein comprises two or three pairs of conservative disulfide bonds, similar motifs of a characteristic motif WIGL and similar motifs of Ca < 2 + >-related sugar binding motifs QPD / EPN and WND, and the protein can be widely combined with polysaccharides on the surfaces of bacteria through preliminary verification, has obvious bacterial binding and bacterial agglutination effects, and can be developed into a natural antibacterial active substance and used for preparing medicines for treating infectious diseases.

Description

technical field [0001] The application belongs to the biological field, and in particular relates to the amphioxus C-type lectin BjCTL4 gene and its application. Background technique [0002] Amphioxus (a representative example such as: Branchiostoma japonicum) belongs to the phylum Chordate and the subphylum cephalochordate, and is the closest living invertebrate to vertebrates. Amphioxus lacks an adaptive immune system similar to that of vertebrates, but it can still survive in a marine environment full of pathogenic microorganisms, mainly relying on its effective and specific immune system. The inductive defense mechanism of the innate immune system is a series of pattern recognition molecules (PRR) that recognize pathogen-associated molecular patterns (PAMPs), and initiate various signaling pathways to control the expression of a series of immune-related genes to achieve the effect of eliminating pathogens. [0003] C-type lectin (CTL) superfamily is a class of proteins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/70C12N1/21A61K38/17A61K48/00A61P31/04C12R1/19
CPCC07K14/461C12N15/70A61K48/005A61K48/0008A61P31/04A61K38/00Y02A50/30
Inventor 黄慧清黄盛丰徐安龙严信宇李军徐立群杨满意徐单单陈素红王霞
Owner GUANGDONG FOOD & DRUG VOCATIONAL COLLEGE
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