Herpes simplex virus type Ⅱ gene recombinant attenuated live vaccine and preparation method thereof

A herpes simplex virus and live attenuated vaccine technology, applied in the field of biomedicine, can solve the problems of difficulty in obtaining pathogenicity, time-consuming and laborious, and achieve the effects of not easy virulence recovery, reducing morbidity, and controlling recurrence and transmission.

Inactive Publication Date: 2015-09-30
郑州金森生物科技工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional live attenuated vaccines are mainly obtained through continuous culture and screening, but this method is time-consuming and laborious, and it is difficult to obtain herpes simplex virus strains with reduced pathogenicity by this method

Method used

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  • Herpes simplex virus type Ⅱ gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus type Ⅱ gene recombinant attenuated live vaccine and preparation method thereof
  • Herpes simplex virus type Ⅱ gene recombinant attenuated live vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Construction of recombinant herpes simplex virus attenuated live vaccine JSH02S

[0028] 1. Construct the homologous recombination shuttle vector pShuttle-02S-GFP.

[0029] (1) With reference to the US2-US5 sequence of the HSV-2 genome whose accession number is NC_001798 on NCBI, design the amplification primers for the left flank sequence of the US2 gene and the right flank sequence of the US5 gene, respectively.

[0030] (2) Primer design for US2 flanking sequences

[0031] A NotI site and three protective bases were added to the 5' end of the upstream primer of the US2 left flank sequence, and a PmeI, AseI site and three protective bases were added to the 5' end of the downstream primer of the US2 left flank sequence. After adding 30 cycles, the target band was recovered.

[0032] (3) Primer design for US5 flanking sequence

[0033] Add PmeI and MluI sites and three protective bases to the 5' end of the upstream primer of the US5 right flank sequence,...

Embodiment 2

[0044] Example 2 Combined application of green and red fluorescent markers to construct recombinant herpes simplex virus attenuated live vaccine JSH02SS

[0045] 1. Use the Axygen plasmid mini-extraction kit to extract the pShuttle-02-RED plasmid according to its instructions.

[0046] 2. Extract the recombinant virus JSH02S genome constructed in the above example, and use liposome 2000 to co-transfect the JSH02S genome and the pShuttle-02-RED plasmid into vero cells.

[0047] 3. Under an inverted fluorescent microscope, aspirate the cells at the CPE that show both red and green fluorescence, and transfer them to an EP tube containing 1ML medium.

[0048] 4. Freeze and thaw the EP tube three times at -80°C-37°C.

[0049] 5. Dilute the virus to an appropriate titer, use the low-melting point agarose covering method for plaque purification, pick out the plaques showing both red and green fluorescence under a fluorescent microscope, and obtain HSV-2 knockout after 5 rounds of pl...

Embodiment 3

[0050] Embodiment 3 Recombinant Herpes Simplex Virus Attenuated Live Vaccine JSH02S and JSH02SS Replication Infection Ability Reduction

[0051] Experimental materials: wild-type HSV-2, JSH02S, JSH02SS, Vero cells, six-well plate.

[0052] Experimental method: 5×10 5 Vero cells were cultured for 24 hours, and the cells in the wells were infected with wild-type HSV-2, JSH02S and JSH02SS according to the multiplicity of infection (MOI) of 0.1 and 1, and the generation of cytopathic effect in the virus-infected wells was observed.

[0053] Results: It is expected that the wild-type HSV-2 will completely produce CPE in about 48 hours to 72 hours, while JSH02S and JSH02SS can produce complete CPE in about 6 to 7 days, indicating that the recombinant strains JSH02S and JSH02SS are more effective than wild-type HSV-2. The ability to infect is significantly reduced.

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Abstract

The invention discloses a herpes simplex virus type 2 (HSV-2) genetic recombinant attenuated live vaccine and a preparation method thereof. US2, US3, US4 and US5 genes in a genome are knocked out. The method comprises the following steps of: separating and identifying vesicle liquid of a patient who suffers from oral herpes and genital herpes and has obvious vesicles to obtain wild type HSV-1 and HSV-2; performing amplifying culture, and extracting a virus complete genome; transfecting Vero cells by using a shuttle vector containing a fluorescent expressed gene and 700bp to 2,000bp homologous flanking sequences on the left and right sides of an HSV gene which is prepared to be knocked out; and picking recombinant virus out under a fluorescent microscope by using one or more fluorescent protein genes as markers, and performing plaque purification to obtain the needed recombinant attenuated live vaccine. The vaccine effectively induces mucosal and humoral immune response of organisms, so that the morbidity of people is reduced; and cellular immune response is excited, so that the infected virus can be cleared, and the relapse and spreading of herpes are controlled.

Description

1. Technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a gene recombinant attenuated live vaccine capable of preventing and treating diseases caused by herpes simplex virus type II infection and a preparation method thereof. 2. Background technology [0002] Herpes simplex virus (HSV) is a virus transmitted through close contact with skin and mucous membranes, including herpes simplex virus type I (abbreviated as HSV-1) and herpes simplex virus type II (abbreviated as HSV-2). Viruses have more than 83% common gene characteristics and more than 50% gene homology. Wherein the infection rate of type I can be as high as more than 80% in the crowd, and long-term dormancy in the sensory ganglion, although most HSV-1 infected persons have no obvious symptoms, but a few infected persons can produce lifelong repeated outbreaks of facial herpes, Highly blinding intraophthalmitis and fatal herpes pneumonia and herpes encepha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/245A61P31/22C12N15/85C12N7/01C12R1/93
Inventor 刘景伟
Owner 郑州金森生物科技工程有限公司
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