Leptospira hemolysin and its code sequence

A leptospira and hemolysin technology, which is applied in the fields of biotechnology and medicine, and can solve problems such as leptospira hemolysin that have not been disclosed.

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

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[0006] However, so f...

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  • Leptospira hemolysin and its code sequence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Bacterial culture

[0074] 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.

[0075] 2) Culture conditions: 30° C., 100 rpm shaking culture for 40 hours.

[0076] 3) Strains: Leptospira interrogans jaundice and haemorrhagic group Lai type Lai strain.

Embodiment 2

[0078] Nucleic acid and amino acid sequences of hemolysin

[0079] Leptospira DNA was extracted by conventional methods to construct a library. Determining the complete sequence of the genome. Based on computer analysis, primers were synthesized, and the extracted Leptospira DNA was used as a template for PCR amplification to obtain the ORF sequence of hemolysin. Sequencing verification again, consistent with the genome sequence. These ORFs encode polypeptides having the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16. The basic information of the eight hemolysins of the present invention is shown in the table below.

[0080] name

[0081] Leptospira hemolysin No.1-8 of the present invention has certain homology with the hemolysin of other bacteria, and has the specific structure domain of hemolysin ( figure 1 ).

Embodiment 3

[0083] Protein

[0084] The Leptospira hemolysin 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 bacteria 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 hemolysin (fusion protein with His-tag) expressed in Escherichia coli was separated and purified by Ni-NTA column to obtain pure Leptospira hemolysin (fusion protein).

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Abstract

The present invention provides a kind of new leptospiral hemolysin, polynucleotide for coding said hemolysin and method for producing said hemolysin by using recombination technique. Said invention also discloses the application of the hemolysin 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 Leptospira hemolysin 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 common. [0004] Althou...

Claims

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Application Information

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IPC IPC(8): C07H21/00C07K14/44C12N15/30C12N15/31C12N15/63C12P21/02G01N33/68
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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