Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Malignant malaria vaccine and preparation method thereof

A technology for microbial strains and silkworms, applied in the field of biomedicine, can solve the problems of low expression, high cost, inability to produce neutralizing antibodies, etc., and achieve the effects of reducing cost, high yield and high application value.

Active Publication Date: 2012-07-11
特菲(天津)生物医药科技有限公司 +1
View PDF3 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the in-depth study of AMA1 molecular biology, people began to develop AMA1 subunit vaccines. At present, the commonly used organisms in biotechnology in the world are Escherichia coli and yeast. The AMA1 protein expressed by E. coli has no immunogenicity and protection, while yeast Expression of AMA1 also fails to produce potent neutralizing antibodies
Mammalian cell expression system (CHO) and other expression systems are effective, but due to the low expression level and the need for a large amount of medium and bovine serum albumin, the cost is high and it is not suitable for production needs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Malignant malaria vaccine and preparation method thereof
  • Malignant malaria vaccine and preparation method thereof
  • Malignant malaria vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Construction of recombinant transfer plasmid pFastBacI-gp64-AMA1

[0029] Using the baculovirus gp64 sequence as a template, the signal peptide (SP) sequence and transmembrane region sequence (TM) of gp64 were amplified by PCR using primers P1, P2, P3, and P4, respectively, and the PCR products passed through Bam H I / EcoR I and xho I / Hind Ⅲ Insert the upstream and downstream ends of the multiple cloning site of the pFastBacI vector by double digestion, and construct the display vector pFstBacI-gp64 (the vector structure is as follows: figure 1 shown). Then, using the Plasmodium falciparum 3D7 standard strain cDNA as a template, primers P5 and P6 were used to amplify the ectodomain of the apical membrane antigen of Plasmodium falciparum AMA1 by PCR, and the PCR product passed Stu I and xho I double restriction digestion was inserted into the surface display vector pFastBacI-gp64, and the recombinant transfer vector pFastBacI-gp64-AMA1 was...

Embodiment 2

[0060] Embodiment 2: Obtaining of Bombyx mori recombinant baculovirus Bmgp64AMA1

[0061] The recombinant transfer plasmid pFastBacI-gp64-AMA1, which was identified as successfully recombined, was transformed into Escherichia coli DH10Bac competent cells (purchased from Invitrogen). (purchased from Shanghai Sangon Biological Co., Ltd.) was screened for blue and white spots, and the white spots were picked after 48 hours of dark culture. After 24 hours of culture, the recombinant baculovirus genome was extracted with isopropanol, and the M13 universal primer was used for PCR identification. The successfully identified recombinant virus genome was transfected into silkworm BmN cells (purchased from Invitrogen) by liposome-mediated method (for the method, refer to the Invitrogen liposome transfection reagent Cellfectin Ⅱ Reagent instructions), and a generation of virus was obtained after onset (microscopic observation) The suspension was stored at 4°C, and the virus genome was ex...

Embodiment 3

[0062] Example 3: Expression of AMA1 fusion protein in 5th instar larvae and pupae of silkworm

[0063] The Bombyx mori recombinant baculovirus Bmgp64AMA1 was used to infect BmN cells at 10 MOI for virus amplification, and the fifth instar larvae or silkworm chrysalis (purchased from Zhejiang Zhongqi Bio-Pharmaceutical Co., Ltd.) After 5-7 days of infection, take larvae or pupal homogenate, centrifuge at a high speed (12000rpm, 30min), take the supernatant, and detect the expression of recombinant protein. Add an equal volume of 2× protein loading buffer (100Mm Tris HCl, 4% SDS, 0.15% bromophenol blue, 10% glycerol) to the supernatant after high-speed centrifugation, heat at 100°C for 10 min, and take 20 μl for SDS-PAGE analysis. The results show that the silkworm recombinant baculovirus has expressed the AMA1 fusion protein, and the protein sequencing results show that its amino acid sequence is shown in SEQ ID NO:4.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to malignant malaria vaccine and a preparation method thereof, which belong to the technical field of biological medicine. A silkworm recombinant baculovirus is constructed, the surface of the recombinant baculovirus is used for showing technical construction and expressing a major antigen anti-mitochondrial antibody 1 (AMA1) extracellular domain of plasmodium falciparum, silkworm chrysalises are used as bio-reactors to generate the recombinant baculovirus, and the recombinant baculovirus is used as original production vaccine of the malaria vaccine. Compared with the traditional vaccine, the malignant malaria vaccine has the advantages of being good in safety, low in production cost, high in yield, easy to operate and suitable for mass production.

Description

technical field [0001] The invention relates to a falciparum malaria vaccine and a preparation method thereof, in particular to the preparation of recombinant plasmids and recombinant viruses in the preparation of falciparum malaria vaccines, and belongs to the technical field of biomedicine. Background technique [0002] Malaria is one of the most serious infectious diseases to humans in the world today. According to the latest WHO estimates, there are 300-500 million cases of acute infection worldwide each year, and more than 1 million people die from this disease, most of whom are children under the age of 5 in sub-Saharan Africa. In recent years, the situation of malaria control has become more severe due to the continuous generation and spread of drug-resistant Plasmodium strains and drug-resistant mosquito vectors. Therefore, the development of an effective malaria vaccine has become an urgent problem to be solved in controlling the morbidity and mortality of malaria....

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N7/01C12N15/30C12N15/866C07K14/445A61K39/015A61P33/06C12R1/93
CPCY02A50/30
Inventor 申俊陈剑清舒特俊张耀洲
Owner 特菲(天津)生物医药科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com