Gene encoding protein of eriocheir sinensis cysteine protease inhibitor Escystatin and application

A cysteine ​​protease, Chinese mitten crab technology, applied in the fields of molecular biology and biochemistry, can solve the problems of economic loss, hindering the development of crustacean breeding industry, frequent occurrence of diseases, etc.

Inactive Publication Date: 2010-10-06
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

However, with the deterioration of breeding scale and breeding conditions, diseases occur more and more frequently, especially infectious diseases caused b

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  • Gene encoding protein of eriocheir sinensis cysteine protease inhibitor Escystatin and application

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Effect test

experiment example 1

[0012] cDNA Cloning of Cysteine ​​Protease Inhibitor Gene of Eriocheir sinensis

[0013] Using T3 primers to sequence the cDNA library of Chinese mitten crab hemocytes, 7535 high-quality EST sequences were obtained. These sequences were spliced ​​into 911 contigs and 2032 singletons, commonly known as Unigenes. The amino acid sequence deduced from one of the spliced ​​sequences has a high homology with the cysteine ​​protease inhibitor protein in higher animals, humans and mice. It is predicted by SMART software that the sequence has a cysteine ​​protease inhibitor protein Like domain, the 3' end of the sequence has a tailing signal (AATAAA) and polyadenylic acid tail (poly A). According to this splicing sequence, forward primer P1 (5'-AATGGCGTTCAACACTTCAC-3') and forward primer P2 (5'-CATCACAATACTATCCTCGCTCAC-3') and adapter dT primer (5'-GGCCACGCGTCGACTAGTACT 17 -3') The 3' end of the cystatin gene is amplified by the rapid amplification technique at the 3' end, and the res...

experiment example 2

[0015] In vitro prokaryotic recombination expression and inhibitory activity determination of the Chinese mitten crab cysteine ​​protease inhibitor gene coding region of the present invention

[0016] 1. Construction of recombinant vector

[0017] The recombinant vector used in the present invention is the pEASY-E1 prokaryotic expression vector of Novagen Company. By PCR technology, primers P5 (5'-ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCGAATT-3') and P6 (5'-GAAATAGGAGATGGGTCGGTCCAAT-3') amplified the coding region fragment of cystatin gene of Chinese mitten crab. The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycles: 94°C denaturation for 30 seconds, 65°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 35 cycles, and finally 72°C extension for 10 minutes. The PCR product was directly connected to the pEASY-E1 prokaryotic expression vector, the connection solution was transformed into Escherichia co...

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Abstract

The invention relates to the technical field of molecular biology and biochemistry, and discloses in-vitro gene recombination and expression technology of an eriocheir sinensis cysteine protease inhibitor (Escystatin) and function identification thereof. The eriocheir sinensis cysteine protease inhibitor is provided with a nucleotide sequence SEQ ID No.1 and a nucleotide sequence SEQ ID No.2. The invention also discloses inhibition effect of the eriocheir sinensis cysteine protease inhibitor on papain. The recombinant protein obtained by using the technology has potential application value in the development of antibacterial medicaments, feed additives and the like.

Description

Technical field: [0001] The invention belongs to the technical fields of molecular biology and biochemistry, and relates to an in vitro recombinant expression technology and activity identification of a Chinese mitten crab cysteine ​​protease inhibitor (EsCystatin) gene. The present invention obtains EsCystatin protein, a cysteine ​​protease inhibitor of Chinese mitten crab, by using in vitro recombinant expression technology. The recombinant protein has a certain inhibitory effect on papain, and has potential application value in the development of antibacterial drugs and feed additives. Background technique: [0002] Cysteine ​​protease inhibitors are protease inhibitors widely present in animal and plant tissues and body fluids. They can form tight complexes with papain, cathepsin and other cysteine ​​proteases to inhibit their activity and protect proteins. The disulfide bonds are not broken by cysteine ​​proteases, preventing protein degradation. Cysteine ​​protein inh...

Claims

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Application Information

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IPC IPC(8): C12N15/15C12N15/70C07K14/81A61K38/57A61P31/00A23K1/16
Inventor 宋林生李凤梅盖雪梅王玲玲邱丽梅
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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