Monoclonal antibodies of avian influenza H5HA antigens

Abstract

The invention discloses monoclonal antibodies of avian influenza H5HA antigens, belonging to the field of biotechnology. The invention provides H5-VN HA genes optimized by the codon of which the sequence is SEQ ID NO.1, and the sequence is used for preparing a monoclonal antibody 3B2IIG8F6 and a monoclonal antibody 4B10IIH4G12 of avian influenza H5-VN HA antigens through DNA immunization and a hybridoma cell CGMCC No.3648 and a hybridoma cell CGMCC No.3649 for secreting the two monoclonal antibodies. The H5-VN HA genes optimized by the codon, which is provided by the invention, can improve the expression of HA genes. In addition, the sequence optimization also enables mRNA to be more stable, and genes can be transcribed and translated more easily. The monoclonal antibodies of H5HA antigens, which are prepared by the invention, provide beneficial tools for research and clinical application of avian influenza viruses.

Description

technical field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody of bird flu H5HA antigen, in particular to a monoclonal antibody of bird flu H5HA antigen prepared by DNA immunization and a hybridoma cell line for producing the antibody. Background technique

[0002] DNA immunization is a new antibody preparation method developed in recent years. DNA immunization overcomes the shortcomings of traditional protein immunization methods, and can more effectively activate the body's humoral and cellular immune responses. Especially in the case where the target protein cannot be obtained by only knowing the DNA code, or the obtained protein has low immunity, it is difficult to obtain specific antibodies by protein immunization, but high-titer specific antibodies can be prepared by DNA immunization, which is Monoclonal antibody preparation provides a simple and easy method. At the same time, the preparation of monoclonal antibodies b...

Examples

Embodiment 1

[0035] The construction of embodiment 1.H5-VN HADNA vaccine

[0036] MacVector 7.2 software was used to analyze Influenza A virus (A / Viet Nam / 1203 / 2004 (H5N1) (according to the published gene bank data), and found that the codons favored by the HA gene derived from A / VietNam1203 / 04 were similar to those of mammalian cells The codons favored by the genes are very different, so the codon-optimized H5-VN HA gene is designed, and the sequence is SEQ ID NO.1. The preferred codons of mammalian cells in the codon-optimized H5-VN HA gene The sub-frequency is higher than that of the wild-type H5-VN HA gene ( figure 1 ). Compared with the wild type, the DNA sequence is changed, but the amino acid sequence of the HA protein is unchanged. The designed sequence was synthesized by Geneart Company in Germany, loaded into plasmid pUC18, and constructed into recombinant plasmid pUC18 / H5-VN. The synthesized sequence was confirmed to be correct by sequencing.

[0037] According to previous r...

Embodiment 2

[0043] Example 2. Cell Transfection

[0044] 293T cells were treated with double-antibody DMEM high-glucose medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator until the logarithmic growth phase, digest with 2.5g / L trypsin, and use 1.0×10 6 Cells (6 mL) were inoculated in a 60 mm culture dish, and transfection was started when they grew to 80% confluence. Cell transfection was carried out according to the PEI transfection method. Take 50 μL of PEI and add it to 930 μL of double-antibody-containing DMEM high-glucose medium, and add 8.0 μg of H5-VN HA recombinant plasmid HA-VN.tPA to the above culture medium, lightly Gently mix, incubate at room temperature for 15min, then add 1ml of PEI / DNA complex to the culture flask and shake gently to mix evenly, meanwhile, transfect cells with pJW4303 empty plasmid as negative control, after 10h, replace without serum containing Double-antibody DMEM medium. Western blot analysis was perf...

Embodiment 3

[0046] Example 3. Establishment of hybridoma cell lines

[0047] Step 1: Animal Immunization

[0048] Seven Balb / c mice were immunized with the HA-VN.tPA vaccine prepared in Example 1. Immunization by intramuscular injection combined with in vivo gene introduction, ensure that the needle insertion depth is 2 mm, inject the vaccine, observe the injection local uplift, and immediately perform in vivo electrotransfection at the injection site with WJ-2002 live gene introduction instrument (technical parameters: voltage 50V, pulse The number of times is 3 times in front and back, the wave width is 60 ms, and the frequency is 30 Hz), and the electrotransfection is effective when the mouse leg muscles shake. DNA immunization was carried out 4 times at week 0, 2, 4, and 8. After the fourth DNA immunization, 293T cells were transfected by intraperitoneal injection of H5-VN.tPA, and each mouse was injected with 5x10 6 -1x10 7 cells.

[0049] Step 2: Cell Fusion

[0050] Four days ...

technical field

[0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody of bird flu H5HA antigen, in particular to a monoclonal antibody of bird flu H5HA antigen prepared by DNA immunization and a hybridoma cell line for producing the antibody. Background technique

[0002] DNA immunization is a new antibody preparation method developed in recent years. DNA immunization overcomes the shortcomings of traditional protein immunization methods, and can more effectively activate the body's humoral and cellular immune responses. Especially in the case where the target protein cannot be obtained by only knowing the DNA code, or the obtained protein has low immunity, it is difficult to obtain specific antibodies by protein immunization, but high-titer specific antibodies can be prepared by DNA immunization, which is Monoclonal antibody preparation provides a simple and easy method. At the same time, the preparation of monoclonal antibodies b...

Examples

Embodiment 1

[0035] The construction of embodiment 1.H5-VN HADNA vaccine

[0036] MacVector 7.2 software was used to analyze Influenza A virus (A / Viet Nam / 1203 / 2004 (H5N1) (according to the published gene bank data), and found that the codons favored by the HA gene derived from A / VietNam1203 / 04 were similar to those of mammalian cells The codons favored by the genes are very different, so the codon-optimized H5-VN HA gene is designed, and the sequence is SEQ ID NO.1. The preferred codons of mammalian cells in the codon-optimized H5-VN HA gene The sub-frequency is higher than that of the wild-type H5-VN HA gene ( figure 1 ). Compared with the wild type, the DNA sequence is changed, but the amino acid sequence of the HA protein is unchanged. The designed sequence was synthesized by Geneart Company in Germany, loaded into plasmid pUC18, and constructed into recombinant plasmid pUC18 / H5-VN. The synthesized sequence was confirmed to be correct by sequencing.

[0037] According to previous r...

Embodiment 2

[0043] Example 2. Cell Transfection

[0044] 293T cells were treated with double-antibody DMEM high-glucose medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultivate in a saturated humidity incubator until the logarithmic growth phase, digest with 2.5g / L trypsin, and use 1.0×10 6 Cells (6 mL) were inoculated in a 60 mm culture dish, and transfection was started when they grew to 80% confluence. Cell transfection was carried out according to the PEI transfection method. Take 50 μL of PEI and add it to 930 μL of double-antibody-containing DMEM high-glucose medium, and add 8.0 μg of H5-VN HA recombinant plasmid HA-VN.tPA to the above culture medium, lightly Gently mix, incubate at room temperature for 15min, then add 1ml of PEI / DNA complex to the culture flask and shake gently to mix evenly, meanwhile, transfect cells with pJW4303 empty plasmid as negative control, after 10h, replace without serum containing Double-antibody DMEM medium. Western blot analysis was perf...

Embodiment 3

[0046] Example 3. Establishment of hybridoma cell lines

[0047] Step 1: Animal Immunization

[0048] Seven Balb / c mice were immunized with the HA-VN.tPA vaccine prepared in Example 1. Immunization by intramuscular injection combined with in vivo gene introduction, ensure that the needle insertion depth is 2 mm, inject the vaccine, observe the injection local uplift, and immediately perform in vivo electrotransfection at the injection site with WJ-2002 live gene introduction instrument (technical parameters: voltage 50V, pulse The number of times is 3 times in front and back, the wave width is 60 ms, and the frequency is 30 Hz), and the electrotransfection is effective when the mouse leg muscles shake. DNA immunization was carried out 4 times at week 0, 2, 4, and 8. After the fourth DNA immunization, 293T cells were transfected by intraperitoneal injection of H5-VN.tPA, and each mouse was injected with 5x10 6 -1x10 7 cells.

[0049] Step 2: Cell Fusion

[0050] Four days ...

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