Epidemic encephalitis B pseudoviruses and preparing method thereof

A pseudovirus, JE technology, applied in the field of JE pseudovirus and its preparation, can solve the problems of low titer of JE pseudovirus, limited application of JE pseudovirus, inability to detect positive fluorescent signals, etc., and achieve convenient quantification. Analysis, good application prospects, simple operation effect

Inactive Publication Date: 2016-05-25
SICHUAN AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few studies on the establishment of pseudovirus vector system for JEV virus, and the titers of JEV pseudovirus obtained after infecting cells are extremely low, which limits the application of JEV pseudovirus
For example, the Japanese encephalitis pseudovirus constructed by Cao Qishu using the E gene of the JEVP3 strain had an infection titer of up to 1.5×10 on 293 cells. 3 TU / ml, no positive fluorescent signal can be detected on BHK21 cells [Cao Qishu, Isolation and identification of porcine Japanese encephalitis virus and construction of E gene pseudovirus [D], Huazhong Agricultural University, 2011]; Leeet et al. used JEVBeijing The Japanese encephalitis pseudovirus constructed with the ME gene of the strain had an infectious titer of 2.05×10 to 293T and BHK-21 cells, respectively. 2 TU / ml and 7.91×10 3 TU / ml【TheprM-independent packaging of pseudotyped Japanese encephalitisvirus[J].Leetal.,2009】; and the lentiviral systems established by the two methods are single-gene reporter systems, either inconvenient to observe the fluorescence of the target protein, or inconvenient for quantitative analysis

Method used

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  • Epidemic encephalitis B pseudoviruses and preparing method thereof
  • Epidemic encephalitis B pseudoviruses and preparing method thereof
  • Epidemic encephalitis B pseudoviruses and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Preparation of the recombinant vector of the present invention

[0042] 1. Design primers

[0043] Amplification primers of the JEVSCYA201201 strain were designed, and the primers could amplify the E gene and the M gene with signal peptide (hereinafter referred to as SPME) of the JEVSCYA201201 strain.

[0044] 2. Amplification and cloning of SPME gene

[0045] Using the frozen purified JEVSCYA201201 strain virus liquid as a template, the virus liquid RNA was extracted according to the instructions of the Beijing Tiangen Total RNA Extraction Kit, and the cDNA of the SPME gene was synthesized by reverse transcription, and the cDNA of the JEVSPME gene was used as a template to design The SPME gene amplification primers of JEV were used to perform PCR amplification of the SPME gene of JEV. The amplification reaction conditions were: 95°C for 2min; 94°C for 1min, 57°C for 30s, 72°C for 120s, 35 cycles; 72°C for 10min.

[0046] After amplification, 5 μL of the PC...

Embodiment 2

[0050] The preparation of embodiment 2 Japanese encephalitis pseudovirus kit of the present invention

[0051] According to the method of Example 1, the recombinant vector pcDNA3.1-SPME was prepared;

[0052] The lentiviral packaging plasmid pCDH-CMV-Luc-copGFP and psPAX2.1 are taken together with the recombinant vector pcDNA3.1-SPME of the present invention to form the kit of the present invention.

Embodiment 3

[0053] Preparation and identification of embodiment 3 Japanese encephalitis pseudovirus of the present invention

[0054] One, the preparation of Japanese encephalitis pseudovirus

[0055] 1. Co-transfection

[0056] The kit prepared by the method in Example 2 was transfected by the cationic liposome method.

[0057] The weight ratio of the plasmids used for transfection is: packaging plasmid pCDH-CMV-Luc–copGFP: helper plasmid psPAX2.1: recombinant plasmid pcDNA3.1-SPME is 4:3:2;

[0058] According to the total weight of the plasmids used: the transfection reagent Lipofectamine3000 was 1 μg: 1.5 μl, and the 293T cells were co-transfected.

[0059] 2. Harvest JE pseudovirus

[0060] Cultivate the host cells for 48 hours, collect the supernatant by centrifugation (centrifugation conditions: 4° C., 8000 g, 10 min), and filter the supernatant with a sterile 0.45 μm filter to obtain the Japanese encephalitis pseudovirus supernatant of the present invention. For the packaging p...

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Abstract

The invention discloses recombinant vectors. The recombinant vectors are recombinant plasmids pcDNA3.1-SPME carried by recombinant bacteria DH5alpha/pcDNA3.1-SPM, the collection number of the recombinant plasmids in the China center for type culture collection (CCTCC) is CCTCC NO:M2015570. The invention further discloses a kit for preparing epidemic encephalitis B pseudoviruses and an application thereof. The invention further discloses a preparing method of the pidemic encephalitis B pseudoviruses and the recombinant bacteria. By means of the constructed recombination vectors pcDNA3.1-SPME, the epidemic encephalitis B pseudoviruses can be efficiently obtained, the titer of the obtained pidemic encephalitis B pseudoviruses can be high, and good application prospects can be achieved.

Description

technical field [0001] The invention relates to a Japanese encephalitis pseudovirus and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Epidemic encephalitis (epidemicencephalitisB), also known as Japanese encephalitis, Japanese encephalitis (JapanesetypeBencephalitis), is a kind of zoonotic insect-borne infectious disease caused by Japanese encephalitis virus (JEV, Japaneseencephalitisvirus), which can cause children Non-suppurative encephalitis and abortion of sows have brought great harm to human health and the breeding industry. [0003] When conducting drug screening and scientific experiments on JE, JEV needs to be used directly. However, due to its strong infectivity and high pathogenicity, JEV is a virus with a hazard level of II, which brings great potential infection risks to researchers. Therefore, it is urgent to develop safe and effective means of JEV research. [0004] Pseudovirus refers to a retrovirus tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N1/21C12R1/93C12R1/19
CPCC07K14/005C12N7/00C12N15/86C12N2740/15021C12N2740/15043C12N2770/36022C12N2770/36052
Inventor 曹三杰文心田刘瀚扬伍锐黄小波文翼平袁磊冯瑶周玉鹏曹玉琴
Owner SICHUAN AGRI UNIV
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