Leptospira vaccine candidate outer membrane protein

A leptospira and outer membrane protein technology, applied in the fields of biotechnology and medicine, can solve the problems of narrow scope of action, insufficient immunity, and limited application of leptospira vaccines.

Inactive Publication Date: 2009-09-30
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But their common disadvantages are that the immunity is not durable enough, the potency is low, the scope of action is narrow, and it needs to be immunized in the epidem

Method used

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  • Leptospira vaccine candidate outer membrane protein
  • Leptospira vaccine candidate outer membrane protein
  • Leptospira vaccine candidate outer membrane protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Cloning, expression and identification of a new vaccine candidate gene LA3744

[0092] The LA3744 gene was cloned and expressed by conventional molecular biology methods, the expression vector was pET28b+, and the expression strain was E.coli BL21-3E. After screening positive clones for Kan resistance, they were added to LB liquid medium and amplified overnight at 37°C and 250rpm. Transfer to LB medium at an inoculum size of 1:100, culture at 37°C for 2.5hrs until the OD is about 0.6, add IPTG to a final concentration of 0.5mM, induce protein expression at 30°C 250rpm for 8hrs. Ultrasonic crush the E.coli BL21-3E cells that have expressed the protein, and collect the precipitate. According to the method provided by Qiagen, the components containing the recombinant protein were collected by Ni-NTA affinity chromatography, and subjected to dialysis and renaturation. The results of PCR amplification products, enzyme digestion identification of recombinant pla...

Embodiment 2

[0094] Example 2 Immunogenicity detection of recombinant proteins

[0095] 400 μg of recombinant protein plus an equal volume of complete Freund’s adjuvant (Sigma Company) was used to immunize 3 kg of New Zealand rabbits. PBS + adjuvant was used as a negative control group. After the initial immunization, 200 μg of recombinant protein plus an equal volume of incomplete Freund’s adjuvant was used to boost once every 15 days , blood was collected on the 10th day after two times of strengthening, and the potency was measured by ELISA. The antibody titer produced by recombinant protein PL40 is greater than 1:16000. , showing good immunogenicity. The immunoreactivity of the recombinant protein was detected by Western-blot, which showed that the prepared polyclonal antibody could effectively bind to the corresponding recombinant protein.

Embodiment 3

[0096] Example 3 Conservative Detection of LA3744 and Its Encoded Protein in Leptospira Endemic Strains in China

[0097]Conservation detection at the nucleic acid level: Using 15 Chinese strains and the total DNA of the double helix as templates, primers were designed for the conserved regions for PCR amplification. Specific amplified bands were produced in 14 strains, while Weissner No specific bands were produced in Leptospira clear water type L 105 strain and non-pathogenic hyperbolic Leptospira ( Figure 5 ).

[0098] Conservation detection at the protein level: PL40 antiserum was used to detect the expression of representative strains of 15 serogroups prevalent in China by Western blot. Corresponding hybridization bands can be detected in 14 strains, and the molecular weight is consistent with the theoretical value, indicating that the protein is expressed, but it is not detected in the L105 strain of Leptospira wesmanii Shimizu type and the non-pathogenic Leptospira...

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PUM

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Abstract

The invention provides a leptospira outer membrane protein capable of serving as a candidate vaccine, a polynucleotide for coding the outer membrane protein and a method for generating the outer membrane protein through a recombinant technique. The outer membrane protein is a useful immunogen for preparing the vaccine. The invention also discloses application of the protein and a coded sequence thereof for preparing the vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology and medicine, and in particular relates to a leptospira outer membrane protein which can be used as a candidate vaccine. Background technique [0002] Leptospira is a slender, curved, helical, and active prokaryotic microorganism belonging to the genus Leptospira in the family Spirocaetalae of the order Spirocheatals. Previously, Leptospira was divided into 2 species: pathogenic Leptospira interrogans (L.interrogans generalized) and saprophytic hyperbolic Leptospira (L.biflexa generalized). Leptospira interrogans can be divided into different serotypes according to the cross-agglutination test, at least 25 serogroups and more than 200 serotypes. There are 18 groups of 75 types in my country, which is the country with the most serotypes found in the world. Leptospira have been classified according to genospecies based on the results of DNA-DNA hybridization in recent years. At present, it is divided...

Claims

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Application Information

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IPC IPC(8): C07K14/20C12N15/31C12N15/63C12N1/21C12N1/19C12N5/10C12P21/02C07K16/12A61K39/02A61P31/04C12R1/01
CPCY02A50/30
Inventor 郭晓奎秦金红赵蔚陈春燕杨宏亮朱泳璋何平
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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