Sturgeon disease-resistant immune protein and preparation method and application thereof

A sturgeon protein technology, applied in the field of sturgeon disease-resistant immune protein and its preparation, to achieve the effects of inhibiting microbial infection, promoting development, and improving immunity

Active Publication Date: 2019-03-08
BEIJING FISHERIES RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been no reports on the gene cloning, expression analysis, preparation of recombinant protein and its pro-lymphocyte proliferation and antibacterial activity of sturgeon interleukin-6

Method used

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  • Sturgeon disease-resistant immune protein and preparation method and application thereof
  • Sturgeon disease-resistant immune protein and preparation method and application thereof
  • Sturgeon disease-resistant immune protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Obtaining of Sturgeon Interleukin-6 Gene and Construction of Expression Vector

[0045] 1. Acquisition of interleukin-6 gene from sturgeon

[0046] In order to study whether conditional stress such as immunogen stimulation or high temperature affects the immune function of sturgeon, the inventor's team carried out liver transcriptome sequencing of sturgeon under different conditions. For the expressed gene, the nucleotide sequence of its cDNA is shown in SEQ ID NO.2, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.1. According to the homologous sequence comparison and protein structure simulation analysis, the gene is speculated The encoded protein is interleukin-6.

[0047] Amplification primers F carrying NcoI and XhoI restriction sites were designed according to the mRNA sequence of the interleukin-6 gene of the above-mentioned targeted sturgeon: (SEQ ID NO.3:C CCATGG CTCCTATTGTAGAATTTTC) and R (SEQ ID NO.4: CCG CTCGAG TCATACAAAT...

Embodiment 2

[0055] Example 2 Induced Expression and Purification of Siberian Acipenser IL-6 Recombinant Protein

[0056] 1. Induced expression of Siberian sturgeon IL-6 recombinant protein

[0057] Inoculate pET30a(+)-AbIL6-BL21 in LB medium containing kanamycin (50 ng / μL), and shake culture at 37°C until OD 600 About 0.6, add 1mM isopropyl-β-D-thiogalactoside, continue to cultivate for 6h, and collect the bacteria by centrifugation. Suspend the cells with 1 / 10 volume of medium in PBS, and add PMSF to a final concentration of 1mM. Under ice bath conditions, lyse the cells by 100W ultrasonic disruption, centrifuge to collect the supernatant and precipitate of the lysate, and perform SDS-PAGE electrophoresis analysis , the results showed that the Siberian sturgeon Il6 recombinant protein existed in the inclusion body ( figure 1 ).

[0058] 2. Purification of Siberian sturgeon IL-6 recombinant protein

[0059] Resuspend the sonicated pET30a(+)-AbIL6-BL21 cell pellet collected in BufferA ...

Embodiment 3

[0070] Example 3 Expression analysis of AbIL6 gene in different tissues of Siberian sturgeon

[0071] The relative expression of IL-6 gene in different tissues of healthy Siberian sturgeon was detected by real-time fluorescent quantitative PCR method, and the tissue distribution of AbIL6 mRNA was studied.

[0072] Specific operation method: randomly select 10 tissues of healthy Siberian sturgeon (about 150 g in weight), which are: gills, eyes, head kidney, spleen, heart, muscle, skin, liver, brain and intestine. Total tissue RNA was extracted by Trizol method and reverse transcribed into cDNA by Takara reverse transcription kit.

[0073] Primers for fluorescent quantitative PCR were designed according to the cDNA sequence of AbIL6, and the primer sequences were as follows:

[0074] SEQ ID NO.5: Abil6F: 5'-CACTGTCAGCGTTGCTGGTTC-3';

[0075] SEQ ID NO.6: Abil6R: 5'-GCCACTCGGCTAAAGATTCCC-3';

[0076] The primer sequence of the internal reference gene 18S rRNA is as follows:

...

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Abstract

The invention relates to sturgeon disease-resistant immune protein and a preparation method and application thereof. The sturgeon disease-resistant immune protein is sturgeon interleukin-6 in an aminoacid sequence shown as SEQ ID NO.1. Expression of the novel sturgeon interleukin-6 is induced by various immune stimulations, and functions of immune tissue cell proliferation promotion, immune molecule expression regulation and bacterial infection inhibition are achieved. The sturgeon interleukin-6 can be used as a disease-resistant preparation such as an immunity enhancement agent to improve sturgeon immunity and effectively prevent and control sturgeon disease damages, and accordingly development of the sturgeon aquaculture industry is promoted.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a sturgeon disease-resistant immune protein and its preparation method and application. Background technique [0002] Sturgeon belongs to cartilage and hard scales, and is the oldest primitive fish in the world. It is called "living fossil" and known as "water panda". Because sturgeon belongs to cartilage and hard scales, between cartilaginous fish and bony fish, it has high academic research value. At the same time, the sturgeon meat is delicious and has high nutritional value, and the sturgeon caviar is known as "black gold" (Jiang Lifan, 1998; Chen Zenglong, 1993). Sturgeon farming has great potential and a broad market prospect. my country's sturgeon aquaculture industry started relatively late but has developed rapidly. After more than 20 years of development, the scale of aquaculture has continued to expand, and the number of aquaculture species has continued to increase. At ...

Claims

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Application Information

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IPC IPC(8): C07K14/54C12N15/24C12N15/85C12N5/10A01K67/027A61K38/17A61P37/04A61P31/00
CPCA01K67/0275A01K2227/40A61K38/00A61P31/00A61P37/04C07K14/5412
Inventor 王晓雯朱华陈静怡张蓉朱建亚刘丽丽
Owner BEIJING FISHERIES RES INST
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