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Recombinant pseudotype baculovirus expressing rabies virus G protein and application

A pseudotyped baculovirus, rabies virus technology, applied in the field of virology and genetic engineering, to achieve the effect of improving expression efficiency

Inactive Publication Date: 2013-05-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the host specificity of the traditional baculovirus, this high-efficiency expression vector is only used to mediate the expression of foreign genes in its host insect cells

Method used

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  • Recombinant pseudotype baculovirus expressing rabies virus G protein and application
  • Recombinant pseudotype baculovirus expressing rabies virus G protein and application
  • Recombinant pseudotype baculovirus expressing rabies virus G protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of transfer plasmid pFastBac-RVG-poly(A)-CMV-EGFP and pFastBac-RVG-poly(A)-CMV-RVG in the present invention

[0035] The construction route diagram of transfer plasmid pFastBac-RVG-poly(A)-CMV-EGFP and pFastBac-RVG-poly(A)-CMV-RVG in the present invention is shown in figure 1 with figure 2 shown.

[0036] 1. Primer design, PCR amplification and gene cloning of poly(A) sequence and EGFP gene

[0037] According to the nucleotide sequence of poly(A) in the vector pCI-neo (Cat.No.E1841, purchased from Promega), design the upstream primer poly(A)-Forward: TTT AGATCT CGACATGATAAGATACA (the underlined part is the Bgl II restriction site); downstream primer poly(A)-Reverse: TTT GGATCC TACCACATTTGTAGAGG (the underlined part is the restriction site of BamH I). The vector pCI-neo was used as a template to amplify the poly(A) sequence. The amplification conditions of the poly(A) sequence were: denatured at 95°C for 3 minutes and then cycled. The cy...

Embodiment 2

[0047] Embodiment 2: the acquisition of recombinant pseudotyped baculovirus vaccine

[0048] The transfer plasmid pFastBac-RVG-poly(A)-CMV-EGFP and pFastBac-RVG-poly(A)-CMV-RVG constructed in Example 1 are used to package the recombinant pseudotyped baculovirus of the present invention, and the detailed steps are as follows:

[0049] 1. Transposition of Transfer Plasmids

[0050] (1) Take 1 ng each of the transfer plasmids pFastBac-RVG-poly(A)-CMV-EGFP and pFastBac-RVG-poly(A)-CMV-RVG, and add to two tubes of DH10Bac competent (Cat.No.12331-013, (purchased from Invitrogen Company), mixed gently, and ice-bathed for 30min.

[0051] (2) Heat shock in 42°C water bath for 45 sec, then ice bath for 2-5 min.

[0052] (3) Add 900 μl of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0), and recover on a shaker at 225 r / min at 37° C. for 4 hours.

[0053] (4) Dilute the above resuscitated bacteria solution with LB medium to 10 -1 、10 -2 、10 -3 , add 1.5% agar powder to ...

Embodiment 3

[0076] Example 3: Recombinant pseudotyped baculovirus BV-RVG / EGFP transduces Hela cells

[0077] 1. Inoculate Hela cells (purchased from the China Center for Type Culture Collection, CCTCC in Wuhan University, Wuhan City, Hubei Province) in a 6-well plate, 9 × 10 5 Cells / well, incubate at 37°C for 1 hour to allow the cells to adhere to the wall.

[0078] 2. Use phosphate buffered saline (PBS, 140mmol NaCl, 2.7mmol KCl, 10mmol NaCl 2 HPO 4 , 1.8 mmol KH 2 PO 4 , pH 7.4) to wash the cells 3 times, insert the recombinant pseudotyped baculovirus BV-RVG / EGFP and wild type baculovirus (Wild type BV, purchased from Invitrogen) into the cells at a dose of MOI=50, at 27°C After 4 hours of exposure, the supernatant was discarded, and DMEM medium containing 5% FBS was added.

[0079] 3. Place cells at 37°C 5% CO 2 After culturing in the incubator for 48 hours, the expression of EGFP was observed under a fluorescent microscope, indicating that the pseudotyped baculovirus modified wi...

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Abstract

The invention belongs to the technical field of virology and gene engineering, and particularly relates to construction, vaccine preparation and application of recombinant pseudotype baculovirus expressing rabies virus main immunogenic glycoprotein (RVG). The recombinant pseudotype baculovirus obtained in the invention contains two expression cassettes of not only an insect cell expression cassette in which RVG expression is driven by a PPH promoter and RVG is displayed on a virion envelop surface, but also a mammal cell expression cassette in which high-efficient expression of RVG in mammal cells is driven by a CMV promoter. A rabies vaccine prepared by the recombinant baculovirus has dual properties of subunit vaccines and live viral vector vaccines. After mouse immunization, the rabies vaccine can stimulate the mouse body to generate high-level rabies virus specific ELISA antibodies, neutralizing antibodies, and IFN-gamma, and has potential vaccine application value. The recombinant baculovirus BV-RVG / RVG is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: V201022. The invention also comprises preparation and application of the recombinant baculovirus vaccine.

Description

technical field [0001] The invention belongs to the technical field of virology and genetic engineering, and in particular relates to the construction of a recombinant pseudotyped baculovirus expressing the main immunogenic glycoprotein (G protein) of rabies virus, and the invention also relates to the construction of the recombinant pseudotyped baculovirus Prepared vaccines and applications. Background technique [0002] Rabies (Rabies) is caused by rabies virus (Rabies virus, RV), which can cause acute encephalitis and peripheral nerve inflammation in humans and mammals. The clinical features of rabies are fear of water, wind, spasm of pharyngeal muscles, and progressive paralysis. Once the onset occurs, the mortality rate is almost 100%. The earliest occurrence of rabies can be traced back to 2300 BC, and it is still prevalent in the world. In Asia, about 35,000 cases of human rabies are reported every year, 94% of which are caused by bites of sick dogs or dogs carrying...

Claims

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Application Information

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IPC IPC(8): C12N15/866A61K39/205A61K48/00A61P31/14C12R1/93
Inventor 方六荣吴群峰肖少波于福来傅振芳陈焕春
Owner HUAZHONG AGRI UNIV
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