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Malignant malaria vaccine and preparation method thereof

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

Active Publication Date: 2014-04-23
特菲(天津)生物医药科技有限公司 +1
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  • 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

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  • Malignant malaria vaccine and preparation method thereof
  • Malignant malaria vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0029] Using the baculovirus gp64 sequence as a template, PCR amplified the signal peptide (SP) sequence and transmembrane sequence (TM) of gp64 with primers P1, P2, P3, and P4, and the PCR product passed Bam H I / EcoR I and Xho I / Hind Ⅲ Double enzyme digestion inserts the upstream and downstream ends of the multiple cloning site of pFastBacI vector to construct the display vector pFstBacI-gp64 (vector structure is as figure 1 Shown). Then, using the cDNA of the standard strain of Plasmodium falciparum 3D7 as a template, PCR was performed with primers P5 and P6 to amplify the AMA1 ectodomain of the apical membrane antigen of Plasmodium falciparum. The PCR product passed Stu I and Xho I double enzyme digestion into the surface display vector pFastBacI-gp64 to construct a recombinant transfer vector pFastBacI-gp64-AMA1. The recombinant transfer vector includes polyhedron promoter (pPol...

Embodiment 2

[0060] Example 2: Obtaining the Bombyx mori recombinant baculovirus Bmgp64AMA1

[0061] Identify the successfully recombined recombinant transfer plasmid pFastBacI-gp64-AMA1 to transform E. coli DH10Bac competent cells (purchased from Invitrogen) on an LB culture plate containing kanamycin, gentamicin, tetracycline, X-gal and IPTG ( (Purchased from Shanghai Shenggong Biological Company) for blue-white spot screening. After 48 hours of dark culture, the white spots were picked. After 24 hours of culture, the recombinant baculovirus genome was extracted with isopropanol, and the M13 universal primers were used for PCR identification. The successfully identified recombinant virus genome was transfected into Bombyx mori BmN cells (purchased from Invitrogen) by liposome-mediated method (refer to the instructions of Invitrogen’s liposome transfection reagent Cellfectin Ⅱ Reagent). After the onset of disease (microscopic observation), a first-generation virus was obtained. The suspensio...

Embodiment 3

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

[0063] The silkworm recombinant baculovirus Bmgp64AMA1 was infected with 10MOI virus to BmN cells for viral amplification, and the 5th-instar larvae or silkworm pupae of silkworm (purchased from Zhejiang Zhongqi Biopharmaceutical Co., Ltd.) were inoculated with 1×10 PFU per acupuncture method. After 5-7 days of infection, the larvae or pupae were collected and centrifuged at a high speed (12000rpm, 30min). The supernatant was taken to 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 it 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 as shown in...

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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 a falciparum malaria vaccine, belonging to the technical field of biomedicine. Background technique [0002] Malaria is one of the most serious infectious diseases in the world today. According to the latest WHO estimates, 300-500 million cases of acute infection occur globally each year, and more than 1 million people die from this disease, most of which are children under 5 years old in sub-Saharan Africa. In recent years, due to the continuous production and spread of drug-resistant Plasmodium strains and drug-resistant mosquito vectors, the situation of malaria control has become more severe. Therefore, the development of an effective malaria vaccine has become an urgent task to be solved to control the incidence and mortality of malaria. [0003] Among the many can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/30C12N15/866C07K14/445A61K39/015A61P33/06C12R1/93
CPCY02A50/30
Inventor 崔立旺张耀洲申俊陈剑清舒特俊
Owner 特菲(天津)生物医药科技有限公司
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