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Engineered saccharomyces cerevisiae capable of efficiently expressing Chinese mitten crab chitin

A technology of Chinese mitten crab and Saccharomyces cerevisiae, applied in fungi, microorganism-based methods, animal feed, etc., can solve the problems of cumbersome renaturation process and low output rate, and achieve healthy growth and enhance resistance. , the effect of efficient expression

Inactive Publication Date: 2016-02-03
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the full-length chitin Crustin gene of Chinese mitten crab (Eriocheirsinensis) has been cloned, and some researchers have expressed prokaryotic expression of the Chinese mitten crab Crustin gene in E. coli, but the E. coli expression system often leads to protein aggregation to form insoluble Active inclusion bodies, complex renaturation process, low output rate and other factors greatly increase the cost of industrial production
However, the study of eukaryotic expression and protein function of Chinese mitten crab Crustin by Saccharomyces cerevisiae has not been reported so far.

Method used

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  • Engineered saccharomyces cerevisiae capable of efficiently expressing Chinese mitten crab chitin
  • Engineered saccharomyces cerevisiae capable of efficiently expressing Chinese mitten crab chitin
  • Engineered saccharomyces cerevisiae capable of efficiently expressing Chinese mitten crab chitin

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: the construction of Saccharomyces cerevisiae genetically engineered bacteria (gene cloning)

[0031]By constructing the high-expression plasmid pHAC181-EsCrustin, the specific method is to extract the tissue RNA of Chinese mitten crab, and the reverse transcription kit reverse-transcribes the RNA into cDNA. Use high-fidelity enzymes to carry out PCR amplification of the target gene fragment EsCrustin, the EsCrustin gene fragment (CDS region, excluding the stop codon, a total of 336bp), and then clone this fragment into the multiple cloning site of the high-expression plasmid pHAC181, and finally correct the DNA sequencing was carried out to verify that the sequence was not mutated, and the recombinant high-expression plasmid pHAC181-EsCrustin was obtained.

Embodiment 2

[0032] Embodiment 2: the construction of Saccharomyces cerevisiae genetically engineered bacteria (homologous recombination)

[0033] Homologous recombination primers were designed based on the constructed recombinant high-expression plasmid pHAC181-EsCrustin, and the plasmid pHAC181-EsCrustin was amplified using the high-fidelity PrimeSTARGXL enzyme, and the successfully amplified long fragment was integrated into the downstream of the GAL1 promoter in the Saccharomyces cerevisiae strain.

[0034] Homologous recombination primers are F1 and R1, and the sequences are shown in SEQ ID NO.3 and SEQ ID NO.4:

[0035] F1: caaatgtaataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaaaacccggatctcaaaATGAAGGTGTGTCTGACCCTCCT

[0036] R1: tatggacgaggtaataaggaaactcagaaccagaatagtggcatgagctctccaatttaacatatttgccattagtgaccCGATGATAAGCTGTCAAACATG

[0037] The specific steps of gene homologous recombination (integration) are:

[0038] 1. Pick a single colony of the activated GAL1-ScRCH1 ...

Embodiment 3

[0052] Example 3: Engineering strain protein expression and WESTERNBLOT detection

[0053] A single colony of the Gal-pHAC181-EsCrustin strain with successful homologous recombination was inoculated in 5ml of SD-LEU medium for overnight culture, and the culture was transferred to 45mLYPG medium the next day, and induced by galactose for 6h (OD detected as 1.2 -1.5) After that, extract the strain protein. The extracted protein was used for WESTERNBLOT detection.

[0054] The specific steps are:

[0055] (1) Extraction of total protein in cells

[0056] 1. Pick a single colony and culture it overnight at 30°C in the desired liquid medium until saturated;

[0057] 2. Take 5 mL of the overnight cultured bacterial solution and add 45 mL of fresh liquid medium, and shake it in a shaker at 30°C for about 6 hours (OD=1.2-1.5) (rotation speed: 220rmp);

[0058] 3. Collect the bacteria at 8000rpm1min, and remove the upper liquid;

[0059] 4. Resuspend the bacteria with pre-cooled d...

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Abstract

The invention discloses engineered saccharomyces cerevisiae capable of efficiently expressing Chinese mitten crab chitin and belongs to the biotechnology field. According to the engineered saccharomyces cerevisiae disclosed by the invention, cDNA of a Chinese mitten crab anti-lipopolysaccharide factor EsCrustin is cloned to a vector plasmid pHAC181, a successfully cloned positive transformant is subjected to sequencing verification, then a homologous recombination method is utilized for integrating a target gene to downstream of a GAL1 promoter in a saccharomyces cerevisiae strain, and under the induction action of galactose, the target protein EsCrustin is efficiently expressed.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae engineering bacterium for efficiently expressing chitin of mitten crab sinensis, belonging to the field of biotechnology. Background technique [0002] Aquaculture is one of the important industries for foreign exchange earning in my country's economy, and fishery has become a growth point of agricultural economy in my country. In recent years, crustacean diseases have become more and more serious, which has brought huge economic losses to the aquaculture industry in my country. [0003] Crustaceans lack the acquired specific immune function, but they have a relatively complete innate non-specific immune system, which can quickly identify and effectively eliminate invading microorganisms. Nonspecific immune mechanisms in crustaceans include the barrier function of the carapace and mucus, phagocytosis by the reticuloendothelial system, and nonspecific humoral molecules. The antimicrobial peptides of ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C08B37/08A23K10/18C12R1/865
Inventor 杜婕蒋伶活
Owner JIANGNAN UNIV
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