Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation

A technology of recombinant baculovirus and recombinant plasmid, applied in application, virus/phage, resistance to vector-borne diseases, etc., can solve the problems of high cost, low expression, rejection and other problems, achieve high yield, high application value, cost reduction effect

Inactive Publication Date: 2015-06-17
特菲(天津)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The organisms commonly used in current biotechnology are various types of yeast sclerotia Escherichia coli, but the Pvs25 protein expressed by Escherichia coli has not been detected to have obvious immunogenicity and immunoprotection in the transmission blocking experiment, while The Pvs25 protein expressed by Pichia pastoris in the yeast can be well expressed, and the obtained antibody can also detect immunogenicity and protection, and has entered the clinical phase I, but when entering the next clinical phase, The body reacts against
The mammalian expression system expressed today can see the effect, but the expression level is very low, the cost is relatively high, and it does not meet the needs of mass production

Method used

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  • Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation
  • Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation
  • Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Construction of recombinant plasmid pFastBacDual-CMV-Pph-SP-TM

[0050] According to the known sequences of CMV, Pph, SP, and TM, add Xba I restriction site and Xho I restriction site between SP and TM nucleotide sequence, add KpnI restriction site before CMV-F, TM-R Afterwards, a HindIII restriction site was added to synthesize the CMV-Pph-SP-TM sequence (as shown in SEQ ID NO: 1), and primers CMV-F (as shown in SEQ ID NO: 2), TM-R (as shown in SEQ ID NO: 2) and TM-R (as shown in shown in SEQ ID NO: 3). Primers are as follows:

[0051] CMV-F5'-CGG GGTACC TAGTTATTAATAGT-3'

[0052] TM-R5'-CCC AAGCTT TTAATATTGTCTAC-3'

[0053] Wherein the underline is the restriction site.

[0054] The target fragment was amplified by PCR using the synthesized CMV-Pph-SP-TM sequence as a template and CMV-F and TM-R as upstream and downstream primers. The reaction system of PCR is:

[0055]

[0056]

[0057] After each component was mixed, put it into a PCR m...

Embodiment 2

[0058] Example 2: Construction of the recombinant transposable plasmid pFstBacDual-CMV-Pph-SP-Pvs25-TM

[0059] The target gene Pvs25 was amplified by PCR using the T-Pvs25-Pvs48 plasmid as a template, and Pvs25-F (as shown in SEQ ID NO: 4) and Pvs25-R (as shown in SEQ ID NO: 5) as upstream and downstream primers. Primers are as follows:

[0060] Pvs25-F5'-CGC TCTAGA ATGGAAGAAAAAAAT-3'

[0061] Pvs25-R5'-CCG CTCGAG AAGGCATACATTT-3'

[0062] Wherein the underline is the restriction site.

[0063] The reaction system of PCR is:

[0064]

[0065]

[0066] After each component was mixed, put it into a PCR machine, PCR reaction parameters: 95°C pre-denaturation for 5 min, 95°C denaturation for 1 min, 56°C annealing for 30 s, 72°C extension for 30 s, 35 cycles, 72°C stop extension for 10 min. After the reaction was completed, the amplified product fragment was identified by electrophoresis, and the target fragment was 414bp in size, and the target fragment was recovere...

Embodiment 3

[0067] Embodiment 3: the acquisition of Bombyx mori recombinant baculovirus BmPvs25

[0068] The recombined transposable plasmid pFastBacDual-CMV-Pph-SP-Pvs25-TM, which was successfully identified for recombination, was transformed into Escherichia coli DH10Bac competent cells containing the baculovirus shuttle vector Bacmid, in the presence of kanamycin, gentamicin, tetracycline, X-gal and IPTG were cultured on the LB culture plate (operated according to the instructions), and the blue and white spots were screened after homologous recombination by transposition. After 48 hours of dark culture, the white spots were picked, and the white spots continued to be treated with tetracycline and kanamycin. , Gentamicin, X-gal and IPTG in the LB culture solution of shake culture 48h after using isopropanol to extract recombinant baculovirus genomic DNA, use M13 universal primer (M13-F as shown in SEQ ID NO: 6 , M13-R as shown in SEQ ID NO: 7), Pvs25-F and Pvs25-R identified the insert...

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Abstract

The invention provides a recombinant plasmid, a recombinant baculovirus prepared by from the recombinant plasmid and application of the virus in preparation of tertian malaria transmission blocking vaccines. The recombinant vector is constructed by inserting a section of recombinant sequence into the pFastBacDual plasmid. The recombinant sequence is formed by: according to CMV, Pph, SP and TM known sequences, adding Xba I enzyme site and Xho I enzyme site between SP and TM sequences, adding KpnI enzyme site in front of CMV-F, and adding HindIII enzyme site after TM-R. The recombinant baculovirus is obtained by: inserting a Pvs25 gene into the recombinant plasmid, conducting homologous recombination with the genome of a shuttle vector Bacmid through transposition, then transfecting a bombyx mori cell, and performing packaging in the bombyx mori cell. The vaccine production method has the characteristics of good safety, low production cost, and easy mass production operation.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a modified bacmid, that is, a recombinant plasmid, and a recombinant baculovirus containing a target gene (Pvs25 gene, a candidate antigen for vivax malaria transmission blocking), prepared by using the recombinant plasmid, The preparation method of the recombinant baculovirus, the fusion protein expressed by the recombinant baculovirus, and the application of the recombinant baculovirus and the fusion protein in the preparation of vivax malaria transmission blocking vaccine. Background technique [0002] Plasmodium vivax is a recurrent acute infectious disease caused by Plasmodium vivax, characterized by repeated attacks at intervals of 48 hours. Concentrated antimalarial drugs available today are also challenged by the emergence of drug-resistant Plasmodium parasites and insecticide-resistant mosquitoes. The preparation of malaria vaccine has become the main mea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N7/01C12N15/30C07K19/00A61K39/015A61P33/06
CPCY02A50/30
Inventor 张耀洲盛稳稳崔立旺郭庆拓舒特俊陈剑清盖其静
Owner 特菲(天津)生物医药科技有限公司
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