A kind of Japanese encephalitis/yellow fever chimeric virus and its preparation method and application

Abstract

Provided is a cDNA clone of a Japanese encephalitis / yellow fever chimeric virus, the nucleotide sequence of which is as shown in SEQ ID NO. 1. Also provided are the Japanese encephalitis / yellow fever chimeric virus and a use thereof, and a vaccine for preventing yellow fever.

Description

technical field

[0001] The invention relates to a chimeric virus, in particular to a Japanese encephalitis / yellow fever chimeric virus and its preparation method and application. Background technique

[0002] Yellow fever is a mosquito-borne infectious disease caused by yellow fever virus infection and mainly occurs in sub-Saharan Africa and tropical South America. The incidence is about 200,000 cases per year, and the mortality rate is as high as 20-50%. 90% of these occur in Africa. Yellow fever virus infection in humans can cause a variety of clinical symptoms ranging from subclinical infection to life-threatening multiple organ failure, jaundice, and hemorrhage, and it is a serious disease for residents living in and travelers to endemic areas. serious public health threat. Since there are currently no effective antiviral drugs against yellow fever, yellow fever vaccination is the only effective way to prevent infection. At present, the commonly used vaccine is live a...

Examples

Embodiment 1

[0031] Embodiment 1 Construction of chimeric virus of the present invention

[0032] 1. Construction and identification of full-length infectious full-length cDNA clone containing JEV vaccine strain SA14-14-2 and recovery of recombinant virus

[0033] 1. Modification of multiple cloning sites of low-copy plasmid pACNR

[0034] Dissolve Oligo-1 (5'-CGCGCCATTAGGCGCCTTATAGATCTAATGTCCGGATTATGGATCCTGATTGATCATTATTTCTAGAATTAC-3', respectively, the underlines are KasI, BglII, BspEI, BamHI, BclI, XbaI recognition sites) and Oligo-2 (5'-TCGAGTAATTCTAGAATAATGATCAATCAGGATCCATAATCCGGAAGGC-AGATA', respectively 1 After the complementary double strands are formed, the 5' and 3' ends form AscI and Xhol enzyme-digested sticky ends respectively, both of which are prepared by Shanghai Yingwei Jieji Biotechnology Co., Ltd.) in 100 μl sterilized double-distilled water, mix 10 μl each, and heat Cool naturally after reaching 100°C, take 2 μl and connect with pACNR double-digested with AscI and Xhol ...

Embodiment 2

[0094] The preparation of embodiment 2 vaccines of the present invention

[0095] The chimeric virus Chimeri-JYF prepared in Example 1 was inoculated into primary hamster kidney cells (PHK cells) according to 0.001-0.1 M.O.I, and after being adsorbed at 36°C±1°C for 1h, added with a final concentration of 0.1% human serum white The protein MEM maintenance solution was cultured at 36°C±1°C for 2-5 days. After the cells showed obvious CPE (cytopathic changes), the supernatant was harvested and filtered to become the vaccine stock solution.

[0096] The vaccine of the present invention is obtained by adding pharmaceutically acceptable adjuvants or auxiliary components to the vaccine stock solution.

[0097] The beneficial effect of the present invention is illustrated below in the mode of experimental example:

technical field

[0001] The invention relates to a chimeric virus, in particular to a Japanese encephalitis / yellow fever chimeric virus and its preparation method and application. Background technique

[0002] Yellow fever is a mosquito-borne infectious disease caused by yellow fever virus infection and mainly occurs in sub-Saharan Africa and tropical South America. The incidence is about 200,000 cases per year, and the mortality rate is as high as 20-50%. 90% of these occur in Africa. Yellow fever virus infection in humans can cause a variety of clinical symptoms ranging from subclinical infection to life-threatening multiple organ failure, jaundice, and hemorrhage, and it is a serious disease for residents living in and travelers to endemic areas. serious public health threat. Since there are currently no effective antiviral drugs against yellow fever, yellow fever vaccination is the only effective way to prevent infection. At present, the commonly used vaccine is live a...

Examples

Embodiment 1

[0031] Embodiment 1 Construction of chimeric virus of the present invention

[0032] 1. Construction and identification of full-length infectious full-length cDNA clone containing JEV vaccine strain SA14-14-2 and recovery of recombinant virus

[0033] 1. Modification of multiple cloning sites of low-copy plasmid pACNR

[0034] Dissolve Oligo-1 (5'-CGCGCCATTAGGCGCCTTATAGATCTAATGTCCGGATTATGGATCCTGATTGATCATTATTTCTAGAATTAC-3', respectively, the underlines are KasI, BglII, BspEI, BamHI, BclI, XbaI recognition sites) and Oligo-2 (5'-TCGAGTAATTCTAGAATAATGATCAATCAGGATCCATAATCCGGAAGGC-AGATA', respectively 1 After the complementary double strands are formed, the 5' and 3' ends form AscI and Xhol enzyme-digested sticky ends respectively, both of which are prepared by Shanghai Yingwei Jieji Biotechnology Co., Ltd.) in 100 μl sterilized double-distilled water, mix 10 μl each, and heat Cool naturally after reaching 100°C, take 2 μl and connect with pACNR double-digested with AscI and Xhol ...

Embodiment 2

[0094] The preparation of embodiment 2 vaccines of the present invention

[0095] The chimeric virus Chimeri-JYF prepared in Example 1 was inoculated into primary hamster kidney cells (PHK cells) according to 0.001-0.1 M.O.I, and after being adsorbed at 36°C±1°C for 1h, added with a final concentration of 0.1% human serum white The protein MEM maintenance solution was cultured at 36°C±1°C for 2-5 days. After the cells showed obvious CPE (cytopathic changes), the supernatant was harvested and filtered to become the vaccine stock solution.

[0096] The vaccine of the present invention is obtained by adding pharmaceutically acceptable adjuvants or auxiliary components to the vaccine stock solution.

[0097] The beneficial effect of the present invention is illustrated below in the mode of experimental example:

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