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Codon-optimized EV71 VP1 gene and nucleic acid vaccine

A technology of codon optimization and nucleic acid vaccine, which is applied in the field of EV71 VP1 gene and its nucleic acid vaccine, can solve the problems of gene cloning difficult to express effectively, stimulating the host immune system, and low immunogenicity

Inactive Publication Date: 2010-10-06
徐娟 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA vaccines use the host's transcription and translation system. Due to the codon preference of natural organisms, it may be difficult for gene clones derived from pathogens to be effectively expressed in heterologous hosts, so they cannot effectively stimulate the host's immune system and make it Produce better immune protection, which is the main reason for the low immunogenicity of current nucleic acid vaccines

Method used

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  • Codon-optimized EV71 VP1 gene and nucleic acid vaccine
  • Codon-optimized EV71 VP1 gene and nucleic acid vaccine
  • Codon-optimized EV71 VP1 gene and nucleic acid vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Construction and Identification of Nucleic Acid Vaccine pJW4303 / VP1

[0062] 1.1 Preparation of Competent Escherichia coli HB101

[0063] 1.1.1 Thaw Escherichia coli HB101 stored in glycerol on ice, inoculate 5ul into 5ml LB medium, place in a constant temperature incubator at 37°C, 180rpm, and shake the bacteria overnight.

[0064] 1.1.2 Under sterile conditions, take 1ml of the overnight culture solution, inoculate it into 200ml of fresh LB medium, place it in a constant temperature incubator at 37°C, 180rpm, and shake for 2 hours.

[0065] 1.1.3 Cool the bacteria in the previous step on ice for 15 minutes.

[0066] 1.1.4 Aseptically dispense into 50ml centrifuge tubes (autoclaved), centrifuge at 2500rpm at 4°C for 15min.

[0067] 1.1.5 Add cold 0.1M Cacl 2 100ml (Cacl 2 have been autoclaved), resuspend the bacteria, and incubate on ice for 30 min.

[0068] 1.1.6 Aseptically dispense into 50ml centrifuge tubes (autoclaved), centrifuge at 2500rpm at 4°...

Embodiment 2

[0105] Example 2. Construction and Identification of Nucleic Acid Vaccine pJW4303 / tPA-VP1

[0106] 2.1 Design PCR primers, respectively: upstream primer SEQ ID NO.5, downstream primer SEQ ID NO.6; using the recombinant plasmid pGA4 / VP1 as a template, amplify the GCTAGC (NheI) and GGATCC (BamHI) VP1 gene fragment of restriction site. The reaction conditions are: 94°C for 4min, 94°C for 1min, 56°C for 1min, 72°C for 1.5min, a total of 25 cycles, and 72°C for 7min.

[0107] 2.2 Take 2ul of the PCR product and run 1% agarose gel electrophoresis for identification.

[0108] 2.3 Identify the correct PCR product with DNA gel recovery kit (E.Z.N.A. TM Gel Extraction Kit (50), OMEGA BIO-Tek Company) recovered and purified 914bp VP1 gene fragment. The specific steps of glue recovery are as 1.4 operation process.

[0109] 2.4 Purified PCR product and vector pJW4303 were double digested with NheI and BamHI respectively; enzyme digestion system:

[0110] 10x buffer(with BSA) 5.0ul...

Embodiment 3

[0137] Construction and Identification of Example 3.pJW4303 / tPA-VP1-tandem Nucleic Acid Vaccine

[0138] 3.1 Design two pairs of PCR primers, both of which use the recombinant plasmid pGA4 / VP1 as a template, use the upstream primer SEQ ID NO.5, and the downstream primer SEQ ID NO.7 to amplify and amplify containing GCTAGC (NheI) and GGTACC (KpnI) VP1 gene fragment 1 of restriction site; Use upstream primer SEQ ID NO.8 and downstream primer SEQ ID NO.7, amplify containing GGTACC (KpnI) and GGATCC (BamHI) VP1 gene fragment 2 of restriction site. The PCR reaction conditions are the same as those in 2.1.

[0139] 3.2 Take 2ul of the PCR product and run 1% agarose gel electrophoresis for identification.

[0140] 3.3 Identify the correct PCR product with DNA gel recovery kit (E.Z.N.A. TM Gel Extraction Kit (50), OMEGABIO-Tek Company) recovered and purified 914bp VP1 gene fragment 1 and 918bp VP1 gene fragment 2. The specific steps of glue recovery are as 1.4 operation pro...

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PUM

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Abstract

The invention belongs to the technical field of medicinal biology and discloses a codon-optimized EV71 VP1 gene and a nucleic acid vaccine. The EV71 VP1 gene has the use preference of a mammal cell and a colon bacillus codon and the sequence of the EV71 VP1 gene is SEQ IDNO.1. Based on the characteristic of the gene, the gene is inserted into a eukaryon expression vector so as to build the nucleic acid vaccine Vector / VP1, Vector / tPA-VP1 and Vector / VP1-tandem. All the three kinds of VP1 DNA vaccines can express VP1 protein better through eukaryotic cell expression and animal immune validation and can be stimulated to generate specific antibodies so as to embody high immunogenicity.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to a codon-optimized EV71 VP1 gene and a nucleic acid vaccine thereof. Background technique [0002] Enterovirus 71 (EV71) is the main pathogen that causes hand, foot and mouth disease. Damage to the central nervous system and respiratory system may occur, causing encephalitis, acute flaccid paralysis, pulmonary edema, and myocarditis. Severe cases develop rapidly and are prone to death or disability. The disease has become a common and frequently-occurring infectious disease among children in many countries and regions, seriously affecting children's health. [0003] The prevalence of EV71 infection is on the rise year by year, the transmission route is complicated, and a pandemic can occur in a short period of time. It is difficult to prevent and control. There is a lack of specific treatment methods and effective preventive measures, but only symptomatic treatment. ...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/79A61K48/00A61K39/00A61P31/14
Inventor 徐娟王世霞黄祖瑚卢山
Owner 徐娟
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