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Recombinant vector, recombinant baculovirus prepared with 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 high cost, low expression amount, 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 with the same and application of virus in preparation of malaria vaccines
  • Recombinant vector, recombinant baculovirus prepared with the same and application of virus in preparation of malaria vaccines
  • Recombinant vector, recombinant baculovirus prepared with the same and application of virus in preparation of malaria vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of recombinant vector pFastBacDual-CMV-Ph-SP-TM

[0053] According to the known sequences of CMV, Ph, SP, and TM, an EcoRI restriction site (GAATTC) and an Xho I restriction site (CTCGAG) were added between the SP and TM nucleotide sequences, and a KpnI restriction site was added before CMV-F Point, add HindIII restriction site after TM-R, 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 (shown in SEQ ID NO: 3). Primers are as follows:

[0054] CMV-F5'-CCC GGTACC TAGTTATTAATAG-3'

[0055] TM-R5'-CCC AAGCTT TTAATATTGTCTAC-3'

[0056] Wherein the underline is the restriction site.

[0057] 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 50μL, and the specific components are: 10×PCRBuffer5μL, 2.5mmol / 5 μL of dNTPs in mL, 1 μL each of CMV-F a...

Embodiment 2

[0060] Example 2: Construction of the recombinant transposable plasmid pFstBacDual-CMV-Ph-SP-Pys25-TM

[0061] Use the Pys25 target gene (as shown in SEQ ID NO: 4) as a template, and use Pys25-F (as shown in SEQ ID NO: 5) and Pys25-R (as shown in SEQ ID NO: 6) as upstream and downstream primers for PCR Amplify the target gene Pys25.

[0062] Pys25-F5'-CG GAATTC ATGAACACATACTAC-3'

[0063] Pys25-R5'-G GAATTC ATGTTGAGCTTCTTTGGC-3'

[0064] Wherein the underline is the restriction site.

[0065] The PCR reaction system is 50 μL, and the specific components are: 10×PCRBuffer 5 μL, 2.5 mmol / mL dNTPs 5 μL, 0.01 nmol / μL Pys25-F and Pys25-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. The PCR reaction parameters were: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 1 minute, 53°C annealing for 30 seconds, 72°C extension for 45 seconds, 30 cycles, and 72°C extension for 10 minutes. After t...

Embodiment 3

[0067] Embodiment 3: the acquisition of Bombyx mori recombinant baculovirus BmPys25

[0068] The recombinant transposable plasmid pFastBacDual-CMV-Ph-SP-Pys25-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 48h after shake culture, extract recombinant baculovirus genomic DNA with isopropanol, use M13 universal primer (M13-F as shown in SEQ ID NO: 7 , M13-R as shown in SEQ ID NO: 8), Pys25-F and Pys25-R identified the insertion...

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Abstract

The invention provides a recombinant vector, a recombinant baculovirus prepared with 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 Plasmodium yoelii Pys25 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 P25 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 Pys25 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. It is estimated that in 2010, there were more than 650,000 ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/866C07K19/00A61K48/00A61K39/015A61P33/06
CPCY02A50/30
Inventor 张耀洲李伟杰崔立旺王小飞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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