L-type lectin of litopenaeus vannamei as well as coding gene and application of L-type lectin

A technology of vanabine and lectin, applied in the field of genetic engineering

Active Publication Date: 2017-11-21
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

L-type lectin is an immune defense factor, and its gene characteri

Method used

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  • L-type lectin of litopenaeus vannamei as well as coding gene and application of L-type lectin
  • L-type lectin of litopenaeus vannamei as well as coding gene and application of L-type lectin
  • L-type lectin of litopenaeus vannamei as well as coding gene and application of L-type lectin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning, sequencing and sequence analysis of the L-type lectin gene lecV of Litopenaeus vannamei

[0032] (1) Primer design

[0033] Part of the L-type lectin gene sequence was obtained according to the transcriptome data of Litopenaeus vannamei in our laboratory, and primers for 5' / 3' RACE were designed according to this part of the sequence (see Table 1).

[0034] (2) Extraction of RNA

[0035]Total RNA was extracted from gill tissue of Litopenaeus vannamei by Trizol method. Operation steps: Put appropriate amount of ultra-low temperature frozen gill tissue blocks into a 1.5mL centrifuge tube, add 500μL of Trizol extract (invitrogen), and extract RNA according to the instructions of Trizol extract. RNA electrophoresis results such as figure 1 shown.

[0036] (3) Synthesis of cDNA, amplifying the cDNA of gene lecV and cloning and sequencing

[0037] 1) 3' RACE

[0038] Using the extracted gill tissue RNA of Litopenaeus vannamei as a template, PrimeScr...

Embodiment 2

[0048] Example 2: Construction of prokaryotic recombinant expression vector pET28b-lecV

[0049] The prokaryotic expression vector pET28b was amplified by primer pET28b-F and primer pET28b-R. Operation steps: The total volume of the PCR reaction system is 50 μL: prokaryotic expression vector pET28b 2 μL, primer pET28b-F 1 μL, primer pET28b-R 1 μL, Primestar DNA polymerase master mix (TaKaRa, China) 25 μL, add ddH 2 O to make up to 50 μL. The reaction conditions were: 98°C for 2.5 min; 35 cycles of denaturation at 98°C for 10 sec, annealing at 56°C for 25 sec, and extension at 72°C for 5 min; and reaction at 72°C for 10 min. The above PCR product was purified using a PCR product purification kit (TaKaRa, China) to obtain a linearized pET28b vector.

[0050] The L-type lectin gene lecV of Litopenaeus vannamei was amplified with primers LectinEx-F and LectinEx-R. Specific operation steps: The total volume of PCR reaction system is 50 μL: cDNA template 2 μL, primer LectinEx-F 1...

Embodiment 3

[0054] Example 3: E.coli BL21 (pET28b-lecV) induced expression and purification of LecV protein

[0055] Take 100 μL of the cultured E.coli BL21 (pET28b-lecV) in 4 mL of liquid LB medium and culture it on a shaker at 37°C until the OD 600 When it reached 0.6, IPTG was added to induce, and the bacterial solution without IPTG was used as a control. The working concentration of IPTG was 0.2mM, and the induction time was 6h.

[0056] 1 mL of samples without IPTG induction and samples induced with IPTG were taken for total protein analysis. The specific steps are: take 1 mL of bacterial liquid and centrifuge at 12,000 rpm for 3 min, discard the supernatant, and add 200 μL of ddH 2 O To resuspend the bacterial solution, take 15 μL of the bacterial solution and add 4 μL of 5×protein loading buffer, heat in boiling water for 4 minutes, and store at -20°C.

[0057] SDS-PAGE electrophoresis detection and analysis of protein samples. Specific steps: Prepare separating gel and stackin...

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Abstract

The invention discloses an L-type lectin of litopenaeus vannamei as well as a coding gene and the application of the L-type lectin. According to the invention, an L-type lectin gene lecV of litopenaeus vannamei is cloned from gill tissues of litopenaeus vannamei and has a nucleotide sequence as shown by 18th-1007th bases of SEQ ID NO. 1; the L-type lectin lecV of litopenaeus vannamei has an amino acid sequence as shown in SEQ ID NO. 2. The L-type lectin lecV of litopenaeus vannamei provided by the invention has functions of agglutinating bacteria and rapidly removing bacteria in shrimps, which indicates that the L-type lectin lecV of litopenaeus vannamei is involved in an immune regulation mechanism of shrimps, thereby promoting the research of immune regulation mechanism of the shrimps, providing a reliable genetic marker for breeding shrimp varieties with disease resistance and stress resistance, and also providing a novel target site for genetic improvement of the shrimps.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an L-type lectin of Litopenaeus vannamei and its coding gene and application. Background technique: [0002] L-type Lectin was first discovered and named in the seeds of legumes. Plant-derived L-lectins are soluble molecules, whereas in animals L-lectins are membrane-bound proteins. Four L-type lectins have been found in humans and other mammals: ERGIC-53, ERGIC-53-like lectins, VIP36, and VIP36-like lectins. These lectins all contain a large extracellular domain and a short cytosolic domain involved in the early protein secretion pathway. The main difference between L-type lectins and other lectins is their tertiary structure. The carbohydrate recognition domains of monomeric or more complex lectins are connected by β-bent or antiparallel β-sheet short loops. Does not contain α-helical structure. These sheets form a roof-like structure, with sugar-bi...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N1/21A61K38/17A61P31/04C12R1/19
CPCA61K38/00C07K14/43509C12N15/70Y02A50/30
Inventor 罗鹏田雨顺陈廷黄文胡超群霍达
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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