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Malaria vaccine and preparation method thereof

A technology of microbial strains and recombinant baculovirus, applied in the field of biomedicine, can solve the problems of high transmission density of malaria, long half-life of drugs, insufficient drug dosage, etc., and achieve the effect of high application value, less pyrogenicity, and cost reduction

Inactive Publication Date: 2012-07-04
特菲(天津)生物医药科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After a long period of use of any drug, its receptors will develop a certain tolerance to it. Due to insufficient drug dosage, incomplete treatment, long half-life of the drug, and high density of malaria transmission, it is possible to cause Plasmodium falciparum to have a certain tolerance to it. Chloroquine resistance presents serious challenges to drug therapy

Method used

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  • Malaria vaccine and preparation method thereof
  • Malaria vaccine and preparation method thereof
  • Malaria vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Recombinant transfer plasmid pFastBacI-gp64-MSP1 19c build

[0042] Using the baculovirus gp64 sequence as a template, primers P1 (as shown in SEQ ID NO: 1), P2 (as shown in SEQ ID NO: 2), P3 (as shown in SEQ ID NO: 3), and P4 ( As shown in SEQ ID NO: 4), the signal peptide (SP) sequence and the transmembrane region sequence (TM) of gp64 were amplified by PCR, and the PCR product was passed through Bam H I. EcoR I and xho I. Hind III (both purchased from Fermentas Company) were double digested and inserted into the upstream and downstream ends of the multiple cloning site of the pFastBacI vector to construct the display vector pFstBacI-gp64. Using Plasmodium falciparum 3D7 standard strain cDNA (gifted by Professor Yang Zhaoqing, Kunming Academy of Sciences, Yunnan) as a template, PCR amplification of malignant The DNA sequence of the 19kD antigenic site of the Plasmodium merozoite surface membrane protein 1 C-terminus, the PCR product passed EcoR ...

Embodiment 2

[0075] Embodiment 2: Bombyx mori recombinant baculovirus BmNPV-gp64-MSP1 19c acquisition and expansion of

[0076]Escherichia coli DH10Bac competent cells (purchased from Invitrogen) were successfully identified by recombinant pFastBacI-gp64-MSP119c, in the presence of kanamycin (Kan), gentamycin (Gen), tetracycline (Tet), X-gal and IPTG (isopropylthio-β-D-galactoside) LB culture plate was used for blue and white spot screening, and the white spot was picked after 48 hours of dark culture, and the genome was extracted with isopropanol after 24 hours of culture, and the M13 universal primer was used and MSP119c primers for PCR identification, the results showed that specific bands appeared between 2500bp~3000bp and 400bp~500bp respectively (see Figure 7 ).

[0077] The successfully identified recombinant virus genome was transfected into silkworm BmN cells by liposome-mediated method (refer to the Invitrogen company's liposome transfection reagent Cellfectin Ⅱ Reagent manual...

Embodiment 3

[0078] Embodiment 3: Bombyx mori recombinant baculovirus BmNPV-gp64-MSP1 19c Expression and identification in silkworm chrysalis

[0079] Select the silkworm chrysalis on the 6th to 9th day after clustering (the first-generation hybrid Jingsong × Haoyue, produced by Zhejiang Zhongqi Biological Pharmaceutical Co., Ltd.), and remove necrotic, septic, and bad silkworm chrysalis to reduce the number of silkworm chrysalis after injection. Bacterial infection rate, after disinfection with 70% infiltration alcohol, inoculate 1~5 μl of the fourth-generation recombinant virus obtained in Example 3 on the intersegmental membrane, place it in an artificial climate box after injection, and protect it at 25°C for 4-5 days, let The recombinant virus was fully propagated, and then the infected silkworm chrysalis were collected, and some of them were used for detection, and the rest were stored at -80°C for later use.

[0080] 10 g of the above-mentioned diseased silkworm chrysalis were resp...

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Abstract

The invention relates to a malaria vaccine and a preparation method thereof and belongs to the technical field of medicaments. A recombinant silkworm baculovirus is constructed, the 19kD segment (MSP119C) at a tail end of a plasmodium falciparum merozoite surface membrane protein 1C is expressed on the surface of the silkworm baculovirus by baculovirus surface display technology, a recombinant baculovirus is generated by using a silkworm pupa as a biological reactor, and the malaria vaccine is prepared from the recombinant baculovirus. Compared with the traditional malaria vaccine production method, the method has the characteristics of high safety, low production cost, high yield, easiness in operation and suitability for mass production.

Description

technical field [0001] The invention relates to a malaria vaccine and a preparation method thereof, in particular to the preparation of recombinant plasmids and recombinant viruses in the preparation of malaria vaccines, and belongs to the technical field of biomedicine. Background technique [0002] According to the statistics of the World Health Organization, there are about 320 million people living in malaria-endemic areas in the world, killing about 1 million people and causing 400 million people to be infected every year. One hundred million U.S. dollars. Due to the emergence of new modes of transmission, such as the mobility of the infected population and blood transmission, the control of malaria epidemics is facing more severe challenges. Children under the age of five account for 92% of malaria-related deaths each year. Therefore, the development of malaria vaccine is imperative and has become an important part of malaria prevention and control. [0003] Plasmod...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/30C12N15/866C07K14/445A61K39/015A61P33/06C12R1/93
CPCY02A50/30
Inventor 吴昕昕陈剑清舒特俊张耀洲
Owner 特菲(天津)生物医药科技有限公司
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