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Recombinant vector, recombinant baculovirus prepared from the same and application of virus in preparation of malaria vaccines

A technology of recombinant baculovirus and recombinant vector, which is applied in the field of biomedical technology and can solve the problems of low expression amount, high cost, unsuitable for production needs and the like

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

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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  • Recombinant vector, recombinant baculovirus prepared from the same and application of virus in preparation of malaria vaccines
  • Recombinant vector, recombinant baculovirus prepared from the same and application of virus in preparation of malaria vaccines
  • Recombinant vector, recombinant baculovirus prepared from the same and application of virus in preparation of malaria vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of recombinant vector pFastBacDual-CMV-Ph-SP-TM

[0052] According to the known sequences of CMV, Ph, SP, TM, EcoR I restriction site (GAATTC) and Xho I restriction site (CTCGAG) are added between SP and TM nucleotide sequences, and KpnI restriction site is added before CMV-F site, after TM-R, add a HindIII restriction site, synthesize CMV-Ph-SP-TM sequence (as shown in SEQ ID NO: 1), and design primer CMV-F (as shown in SEQ ID NO: 2) , TM-R (as shown in SEQ ID NO: 3). Primers are as follows:

[0053] CMV-F 5'-CCC GGTACC TAGTTATTAATAG-3'

[0054] TM-R 5'-CCC AAGCTT TTAATATTGTCTAC-3'

[0055] Wherein the underline is the restriction site.

[0056] Use the synthesized CMV-Ph-SP-TM sequence as a template, and use CMV-F and TM-R as upstream and downstream primers to amplify the target fragment by PCR. The PCR reaction system is 50 μL, and the specific components are: 10×PCR Buffer 5 μL, 2.5 mmol 5 μL of dNTPs / mL, 1 μL of 0.01 nmol / μL CMV-F a...

Embodiment 2

[0059] Example 2: Construction of the recombinant transposable plasmid pFstBacDual-CMV-Ph-SP-Pys48-TM

[0060] Using the Pys48 target gene (as shown in SEQ ID NO: 4) as a template, using Pys48-F (as shown in SEQ ID NO: 5) and Pys48-R (as shown in SEQ ID NO: 6) as upstream and downstream primers for PCR Amplify the target gene Pys48.

[0061] Pys48-F 5'-CG GAATTC ATGAACACATACTAC-3'

[0062] Pys48-R 5'-G GAATTC ATGTTGAGCTTCTTTGGC-3'

[0063] Wherein the underline is the restriction site.

[0064] The PCR reaction system is 50 μL, and the specific components are: 10×PCR Buffer 5 μL, 2.5 mmol / mL dNTPs 5 μL, 0.01 nmol / μL Pys48-F and Pys48-R 1 μL each, template 2 μL, TaqDNA polymerase 2 μL, ddH 2 O34 μL. 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, 53°C annealing for 30 s, 72°C extension for 45 s, 30 cycles, 72°C extension for 10 min. After the reaction was completed, the am...

Embodiment 3

[0066] Embodiment 3: the acquisition of Bombyx mori recombinant baculovirus BmPys48

[0067] The recombinant transposable plasmid pFastBacDual-CMV-Ph-SP-Pys48-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 LB culture plates (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 48h after shake culture, use isopropanol to extract recombinant baculovirus genomic DNA, use M13 universal primer (M13-F as shown in SEQ ID NO: 7 , M13-R as shown in SEQ ID NO: 8), Pys48-F and Pys48-R identified the insertion ...

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Abstract

The invention provides a recombinant vector, a recombinant baculovirus prepared by from the vector and application of the virus in preparation of malaria vaccines. The recombinant vector is constructed by inserting a section of recombinant sequence into the pFastBacDual vector. The recombinant sequence is formed by: according to CMV, Ph, SP and TM sequences, adding EcoR I enzyme site and Xho I enzyme site between SP and TM, 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 Plasmodium yoelii Pys48 antigen gene into the recombinant vector, 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. Through simple separation and purification of the recombinant baculovirus, an antibody with good titer can be prepared, thus providing a reference for study of P48 malaria vaccines.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a modified baculovirus vector, that is, a recombinant vector. A recombinant baculovirus containing a target gene (Plasmodium yoelii surface protein Pys48 gene) prepared by using the vector, the recombinant baculovirus The preparation method of the baculovirus, the fusion protein expressed by the recombinant baculovirus, and the application of the recombinant baculovirus and the fusion protein in the preparation of malaria vaccine. Background technique [0002] Malaria transmitted by vector mosquitoes is an infectious disease that seriously threatens human life and health. According to the World Malaria Report 2011 published by WHO in 2011, in 2010, a total of 216 million medical records were found in 106 malaria-endemic countries and regions around the world, of which 86% were children under the age of 5. The research and development of malaria vaccines is one of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/866C07K19/00A61K48/00A61K39/015A61P33/06
CPCY02A50/30
Inventor 崔立旺李伟杰张耀洲盛稳稳闫晶晶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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