Codon-optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof

A technology of codon optimization and nucleic acid vaccine, which is applied in the field of H3HA/XJ3-07 gene and its nucleic acid vaccine, can solve the problems of gene cloning difficult to express effectively, stimulating host immune system, and low immunogenicity

Inactive Publication Date: 2010-11-17
王世霞 +4
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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 H3HA/XJ3-07 gene and nucleic acid vaccine thereof
  • Codon-optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof
  • Codon-optimized H3HA/XJ3-07 gene and nucleic acid vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Construction and Identification of Nucleic Acid Vaccine pJW4303 / H3HA

[0041] 1.1 Preparation of Competent Escherichia coli HB101

[0042] 1.1.1 Thaw Escherichia coli HB101 preserved in glycerol on ice, inoculate 5ul into 5ml LB solution, place in a constant temperature incubator at 37°C, 180rpm, and shake the bacteria overnight.

[0043] 1.1.2 Under sterile conditions, take 1ml of the overnight culture solution, inoculate it into 200ml of fresh LB solution, place it in a constant temperature incubator at 37°C, 180rpm, and shake for 2 hours.

[0044] 1.1.3 Cool the bacteria in the previous step on ice for 15 minutes.

[0045] 1.1.4 Aseptically dispense into 50ml centrifuge tubes (autoclaved), centrifuge at 2500rpm at 4°C for 15min.

[0046] 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.

[0047] 1.1.6 Aseptically dispense into 50ml centrifuge tubes (autoclaved), centrifuge at 2500r...

Embodiment 2

[0085] Example 2. Construction and Identification of Nucleic Acid Vaccine pJW4303 / H3HA-tPA

[0086] 2.1 Design PCR primers, respectively: upstream primer SEQ ID N0.4, downstream primer SEQ ID N0.5; use the recombinant plasmid pGA15 / H3HA as a template to amplify the GCTAGC (NheI) and GGATCC (BamHI) H3HA gene fragment (SEQ ID NO.2) that does not contain the natural signal peptide gene at the 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.

[0087] 2.2 Take 2ul of the PCR product and run 1% agarose gel electrophoresis for identification.

[0088] 2.3 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 H3HA gene fragments. The specific steps of glue recovery are as 1.4 operation process.

[0089] 2.4 Purified PCR product and vector pJW4303 were double digested with NheI and BamHI resp...

Embodiment 3

[0117] See the physical map of pJW4303 / H3HA-tPA nucleic acid vaccine plasmid figure 2 b. The sequence located between the NheI and BamHI endonuclease sites is the H3HA gene. For enzyme digestion results, see Figure 4 Lane1, lane5, Lane6, lane7. After Nhe+BamHI double digestion, the target gene fragment H3HA-tPA was released, the length of which was slightly smaller than 1700bp, as expected, and the pJW4303 / H3HA-tPA nucleic acid vaccine was successfully constructed. Example 3. Construction and Identification of pJW4303 / H3HA-dTM Nucleic Acid Vaccine

[0118] 3.1 Design two pairs of PCR primers, both using the recombinant plasmid Pga15 / H3HA as a template, using the upstream primer SEQ ID NO.4 and the downstream primer SEQ ID NO.6 to amplify the GCTAGC (NheI) and GGATCC H3HA gene fragment (SEQ ID NO.3) of (BamHI) restriction site. The PCR reaction conditions are the same as those in 2.1. The gene fragment is a gene fragment that only encodes the extracellular domain part...

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Abstract

The invention belongs to the technical field of medical biology, and relates to a codon-optimized H3HA / XJ3-07 gene and a nucleic acid vaccine thereof. In the invention, according to an HA protein sequence of an equine A-type influenza virus A / Equine / Xinjiang / 3 / 07 (H3N8), an H3HA / XJ3-07 gene segment which can express a corresponding protein is chemically synthesized after codon optimization, and the segment is cloned into a nucleic acid vaccine vector pJW4303 to construct an equine influenza H3HA nucleic acid vaccine; and further, HA genes are modified, an H3-XJ HA natural signal peptide is replaced by a human tPA signal peptide or the gene segments of part of the HA genes are removed to make the HA genes only express an extracellular domain of the HA protein. The experimental result shows that three H3HA nucleic acid vaccines can be expressed in a 293T cell with high efficiency, and can induce the generation of specific anti-H3HA antibodies in New Zealand white rabbits.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and relates to a codon-optimized H3HA / XJ3-07 gene and a nucleic acid vaccine thereof. Background technique [0002] In recent years, influenza A viruses have been widely prevalent in different species, such as highly pathogenic avian influenza H5N1, novel swine-derived H1N1 influenza, and so on. Influenza A also occurs frequently in equines. According to the difference of hemagglutinin (HA) gene and neuraminidase (neuraminidase, NA) gene of influenza virus in the world, HA is divided into 16 subtypes, and NA is divided into 9 subtypes. Among them, equine influenza viruses include H7N7 and H3N8 subtypes. In the past 30 years, the H7N7 equine influenza virus has not been isolated again, and the H3N8 equine influenza often breaks out in the world, suggesting that the existing vaccines cannot effectively control the epidemic of the virus. [0003] Studies have shown that H3N8 equine i...

Claims

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Application Information

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IPC IPC(8): C12N15/44C12N15/79A61K48/00A61K39/00A61P31/16
Inventor 王世霞周建华许莹黄祖瑚卢山
Owner 王世霞
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