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Leptospira pathogenic related protein and its code squence

A leptospira and related protein technology, applied in the fields of biotechnology and medicine, can solve the problems of undisclosed leptospira pathogenic related proteins

Inactive Publication Date: 2003-09-24
中国科学院上海生物工程研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, so far, no Leptospira pathogenicity-related proteins have been disclosed

Method used

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  • Leptospira pathogenic related protein and its code squence
  • Leptospira pathogenic related protein and its code squence
  • Leptospira pathogenic related protein and its code squence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Bacterial culture

[0064] 1) Culture medium: use EMJH liquid medium to prepare 1000 ml of 5× EMJH medium, and weigh 50 g of BSA. Anhydrous sodium acetate 0.5g, sodium pyruvate 1g, ferrous sulfate 0.25g, 1.5% magnesium chloride 3.5ml, 0.4% zinc sulfate 5ml, 0.15 copper sulfate 1ml, disodium hydrogen phosphate 12.6g, potassium dihydrogen phosphate 1.5 g, sodium chloride 5g, ammonium chloride 1.25g, 0.05% VB 12 2ml, VB 1 0.025g, adjust the pH to 7.2-7.6, set the volume to 1000ml, and sterilize by filtration through nitrocellulose membranes with 0.7, 0.45, and 0.2nm pore sizes, respectively. When used, dilute it 5 times with sterile water.

[0065] 2) Culture conditions: 30° C., 100 rpm shaking culture for 40 hours.

[0066] 3) Strains: Leptospira interrogans jaundice and haemorrhagic group Lai type Lai strain.

Embodiment 2

[0068] Nucleic acid and amino acid sequences of pathogenicity-related proteins

[0069] Leptospira DNA was extracted by conventional methods to construct a library. Determining the complete sequence of the genome. According to the results of computer analysis, primers were synthesized, and the extracted Leptospira DNA was used as a template for PCR amplification to obtain the ORF sequence of the pathogenicity-related protein. Sequencing verification again, consistent with the genome sequence. These ORF codes have SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 , 46, 48, 50, 52, 54 amino acid sequences shown in the polypeptide. The basic information of the eight pathogenicity-related proteins of the present invention is shown in the table below.

[0070] name

ORF sequence SEQ

ID NO:

Length (bp)

protein sequence

SEQ ID NO:

length

(aa)

Signal

peptide *

collagenase ...

Embodiment 3

[0076] Protein

[0077] The Leptospira pathogenicity-related protein gene was cloned into the fusion expression vector pET28b (Novagene Company) by conventional genetic engineering method, and Escherichia coli BL21 (DE3) was selected as the recipient bacterium for fusion expression. After fermentation under conventional conditions, the recombinant bacterial protein was extracted, and then subjected to SDS-PAGE electrophoresis, and the molecular weight was basically the same as the predicted value. The pathogenicity-related protein (fusion protein with His-tag) expressed in Escherichia coli was separated and purified by Ni-NTA column to obtain pure Leptospira pathogenicity-related protein (fusion protein).

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PUM

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Abstract

The present invention provides a kind of new leptospiral pathogen-related protein, polynucleotide for coding said pathogen-related protein and method for producing said pathogen-related protein by utilizing recombination technique. Said invention also discloses the application of said pathogen-related protein and its code sequence.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, in particular, the invention relates to a new polynucleotide encoding a leptospira pathogenicity-related protein and a polypeptide encoded by the polynucleotide. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. Background technique [0002] Leptospirosis is a widely distributed zoonotic disease, mainly distributed in southern China, Southeast Asia, South Asia and other developing countries and regions. In China, the disease was listed as a legal infectious disease in 1995. From 1955 to 1993, the annual average incidence rate was 7 / 100,000, and the annual average mortality rate accounted for 1.02% of the number of cases. [0003] The prevalent flora of leptospirosis in China is dominated by jaundice-haemorrhagic group and Pomona group. There are 15 serotypes in the jaundice hemorrhagic group, among which the Lai type is the most co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00C07K14/44C07K16/20C12N15/30C12N15/63C12N15/64C12P21/02
Inventor 任双喜傅刚曾嵘蒋秀高张怡轩赵国屏陈竺缪有刚徐海郭小奎
Owner 中国科学院上海生物工程研究中心
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