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Hdiv-SARP19-I1 gene and antibacterial application of recombination protein of Hdiv-SARP19-I1 gene

A hdiv-sarp19-i1, recombinant protein technology, applied in the field of genetic engineering

Inactive Publication Date: 2016-07-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The function of SARP19 protein or its homologous protein has not been reported so far

Method used

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  • Hdiv-SARP19-I1 gene and antibacterial application of recombination protein of Hdiv-SARP19-I1 gene
  • Hdiv-SARP19-I1 gene and antibacterial application of recombination protein of Hdiv-SARP19-I1 gene
  • Hdiv-SARP19-I1 gene and antibacterial application of recombination protein of Hdiv-SARP19-I1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Hdiv-SARP19-I1 gene sequence and its optimization

[0056] The Hdiv-SARP19-I1 gene sequence was optimized according to the Hdiv-SARP19-I1 gene sequence (SEQ ID No.1) in the transcriptome of variegated abalone larvae and the codon preference of Pichia pastoris. In order to facilitate purification, a histidine purification tag (6*Histag) and an enterokinase tag were added to the N-terminus of the protein; in order to facilitate cloning, EcoRI and NotI restriction sites were introduced at the 5' and 3' ends, respectively. The optimized gene sequence is shown in SEQIDNo.2, which was synthesized by Shanghai Ruichu Biotechnology Co., Ltd. Among them, the restriction endonuclease EcoRI cutting site, histidine purification tag and enterokinase tag were introduced at bases 1 to 42, and Hdiv-SARP19- In the coding region of the I1 protein, 451-453 are stop codons, and 454-461 are restriction endonuclease NotI cleavage sites.

Embodiment 2

[0057] Embodiment 2: Construction of expression vector

[0058] Pichia pastoris GS115 strain and expression vector pPIC9.0K were purchased from Shanghai Ruichu Biotechnology Co., Ltd. The synthesized target gene and vector pPIC9.0K were double-digested with restriction endonucleases EcoRI and NotI, and the digested products were subjected to agarose electrophoresis and the DNA fragments were recovered and ligated with a gel recovery kit. Specifically: add 5 μL of digested product of the target gene, 7 μL of digested product of pPIC9.0K vector, 1 μL of T4 DNA ligase, 2 μL of 10×T4 DNA ligase buffer, add ddH2O to 20 μL in a sterile EP tube, mix well, and keep at 16°C Ligation for 12 hours; add 10 μL of the ligation product to 90 μL of competent Escherichia coli TOP10, mix well, heat shock at 42°C for 90 s for transformation, add 400 μL of LB liquid medium, shake and incubate at 37°C for 1 hour, and spread the bacterial liquid on LB solids containing 25 μg / mL Zeocin Incubate ove...

Embodiment 3

[0059] Example 3: Transformation and strain screening

[0060] Linearize 5-10 μg of the recombinant plasmid pPIC9.0K-SARP19 using the restriction endonuclease KpnI, recover the linearized pPIC9.0K-SARP19 with a PCR product purification kit, add it to 90 μL of competent Pichia pastoris, mix well, After ice-bath for 5 minutes, electroshock transformation, add 1 mL of sorbitol, place at 30°C for 2 hours, spread the bacterial solution on YPD solid medium containing 100 μg / mL Zeocin, and incubate at 30°C for 2 to 3 days. Select the colony on the transformation plate and culture it upside down on the YPD plate containing geneticin (G418) at a concentration of 0, 0.5 mg / mL, and 2 mg / mL at 30°C for 72 hours, and select the strain that grows well at 2 mg / mL as high copy strain. Streak the high-copy strains on MD and MM agar plates respectively, and observe after culturing at 30°C for 2 days. Mut+ transformants grow normally on both plates.

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Abstract

The invention provides an Hdiv-SARP19-I1 gene and antibacterial application of recombination protein of the Hdiv-SARP19-I1 gene, and relates to genetic engineering. A nucleic acid sequence of a protein encoding region of the Hdiv-SARP19-I1 gene is as shown in a sequence table SEQ ID No.1, the nucleic acid sequence of a sequence of the Hdiv-SARP19-I1 gene is as shown in a sequence table SEQ ID No.2, and the amino acid sequence of the protein sequence of Hdiv-SARP19-I1 is as shown in a sequence table SEQ ID No.3. The amino acid sequence of recombination protein of the Hdiv-SARP19-I1 expressed by pichia pastoris is as shown in a sequence table SEQ ID No.4. The recombination protein of the Hdiv-SARP19-I1 gene can be applied for preparation of protein medicines of antagonism Klebsiella pneumoniae and bactericides of the antagonism Klebsiella pneumoniae.

Description

technical field [0001] The invention relates to the field of genetic engineering in biotechnology, in particular to the antibacterial application of a gene Hdiv-SARP19-I1 and its recombinant protein. Background technique [0002] The Hdiv-SARP19-I1 gene was screened from the transcriptome sequence data of variegated abalone larvae [1] , the length of its coding region is 453bp. The protein encoded by it is similar to the SARP19 protein of corn snail (Littorinalittorea) [2] High similarity, so named Hdiv-SARP19-I1 gene [3] . During the development of variegated abalone larvae, there is an attachment metamorphosis stage, during which the larval tissues adapted to the planktonic disappear, and the larval tissues adapted to the bottom dwelling are formed, and the tissues and organs formed here include the digestive system [4] . Preliminary experiments proved that the Hdiv-SARP19-I1 gene was significantly expressed at a high level in the newly developed digestive glands. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C07K14/435A61K38/17A61P31/04
CPCA61K38/00C07K14/43504C12N15/81C12N2800/102Y02A50/30
Inventor 陈军柯才焕林泽丁少雄
Owner XIAMEN UNIV
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