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Amphioxus c-type lectin bjctl4 gene and its application

A carrier and nucleic acid technology, applied in the fields of application, gene therapy, genetic engineering, etc., can solve problems such as the lack of adaptive immune system in amphioxus

Active Publication Date: 2022-07-08
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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  • Amphioxus c-type lectin bjctl4 gene and its application
  • Amphioxus c-type lectin bjctl4 gene and its application
  • Amphioxus c-type lectin bjctl4 gene and its application

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Effect test

Embodiment 1

[0047] The general method and step of embodiment 1

[0048] cDNA analysis method

[0049] Alignment analysis was done with ClustalX (1.83) software and Gene Doc software, and phylogenetics analysis was basically done with 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's 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, RNA dephosphorylation RACE, decap reaction, RNA oligo ligation, and mRNA reverse transcription were performed to synthesize a cDNA strand. Takara's LA Taq polymerase was used for PCR amplification according to the reaction system of GeneRacerTM Kit. The ORF box gene was amplified using Takara's LATaq polymerase.

[0052] Gene fragment amp...

Embodiment 2

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

[0098] Molecules containing CTLD were found in the Qingdao amphioxus digestive tract cDNA library. 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, which was 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. CTLDs are underlined. "WIGL" and the mannose / glucose recognition site (QPD / EPN) are boxed.

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

Embodiment 3

[0105] Example 3 Sequence analysis of CTLDs of BjCTL4

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

[0107] Sequence analysis of the protein encoded by BjCTL4 found that the molecule contains three pairs of cysteines, belonging to secreted long-loop CTLs. Their primitive and simple structures already possess the characteristics of most traditional CTL molecules, including conserved cysteines and cysteines. WIGL motif, as well as characteristic motifs EPN / QPD and WND motifs (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 s...

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Abstract

The present application belongs to the field of biology, and provides amphioxus C-type lectin BjCTL4 gene and application thereof. The protein encoded by the C-type lectin BjCTL4 gene discovered for the first time in this application, its CTL-like domain has the basic features of traditional CTL, including two or three pairs of conserved disulfide bonds, a motif similar to the characteristic motif WIGL, and with Ca 2+ Similar motifs of related sugar-binding motifs QPD / EPN and WND have been preliminarily verified to be able to extensively bind polysaccharides on the surface of bacteria, and have obvious bacterial binding and bacterial agglutination, which can be developed into natural antibacterial active substances and preparations. Medicines to treat infectious diseases.

Description

technical field [0001] The present application belongs to the field of biology, and specifically relates to the amphioxus C-type lectin BjCTL4 gene and its application. Background technique [0002] Amphioxus (representative example: Qingdao Amphioxus (Branchiostoma japonicum)), belonging to Chordate (Chordate), cephalochordate (cephalochordate), is the most closely related invertebrate to vertebrates. Amphioxus lacks a vertebrate-like adaptive immune system, but still can survive in a marine environment full of pathogenic microorganisms, mainly relying on its efficient and specific immune system. The inductive defense mechanism of the innate immune system is that a series of pattern recognition molecules (PRRs) 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] The C-type lectin (CTL) superfamily is a class of...

Claims

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Application Information

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Patent Type & Authority Patents(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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