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Protein D and HER2 fusion protein and preparing method and application thereof

A technology of fusion protein and protein, which is applied in the fields of biology and medicine, can solve problems such as the impact of vaccine immunity, and achieve the effect of stimulating the body's immune response and delaying the growth rate

Inactive Publication Date: 2016-01-20
重庆科润生物医药研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The downside is that CRM 197 This vector is also used in the Hib and meningitis conjugate vaccine, therefore, the same vector may have a negative impact on the immunity of the vaccine

Method used

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  • Protein D and HER2 fusion protein and preparing method and application thereof
  • Protein D and HER2 fusion protein and preparing method and application thereof
  • Protein D and HER2 fusion protein and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of recombinant PD-HER2 fusion protein therapeutic vaccine engineering bacteria

[0049] 1. Construction of pET30-a(+)-PD-HER2 / BL21(DE3) engineering bacteria

[0050] The PD protein derived from Hi (GenBank accession number: CAA84716) was selected, with a full length of 364 amino acids, as shown in SEQ ID NO:1. The 19-127 amino acids of PD protein are selected as the adjuvant protein, as shown in SEQ ID NO:2. Tumor-associated antigen human HER2 (GenBank accession number: AAA75493.1) has a full length of 1255 amino acids, as shown in SEQ ID NO:3. The HER2 antigen extracellular region fragment (41-560aa) and the HER2 epitope peptide fragment (774-788aa) are selected as vaccine molecular antigens. The amino acid sequence of the HER2 antigen extracellular region fragment (41-560aa) is shown in SEQ ID NO:4, and the HER2 epitope peptide fragment (774-788aa) is shown in SEQ ID NO:5.

[0051] The therapeutic vaccine molecule of the recombinant PD-HER2...

Embodiment 2

[0057] Example 2 Fermentation of recombinant PD-HER2 fusion protein engineering bacteria

[0058]The pET30-a(+)-PD-HER2 / BL21(DE3) engineering bacteria constructed in the above Example 1 were streaked LB plates (kan100mg / L) and cultured in a 37°C constant temperature incubator (Bo Xun, Shanghai) for about 16- 18h, until a single colony grows. A single colony of engineered bacteria was picked and inoculated into 20ml of LB medium (kan100mg / L), and cultured at 37°C and 230rpm for 8h. 0.1% was transferred to 250ml LB (kan100mg / L), 1L Erlenmeyer flask, cultured at 37°C, 230rpm for 13h. Four bottles were cultured in parallel, 1000ml of bacterial solution was prepared, and 5% was inoculated into the medium of the upper tank of the fermenter NLF-2220L. Before inoculation, the pH was adjusted to 7.0 with ammonia water, and the temperature during the fermentation process was controlled at 36°C. The pH value and dissolved oxygen of the culture medium were controlled by adding ammonia...

Embodiment 3

[0060] Example 3 Separation and purification of recombinant PD-HER2 fusion protein

[0061] 1. Inclusion body washing

[0062] (1) First wash

[0063] Take the precipitate collected in Example 2, the main component is inclusion body (IB). Wash with washing buffer (20mM Tris-HCl, 2M urea (Sinopharm, Shanghai), 1% TritonX-100 (sigma, USA), 5mM EDTA (Sinopharm, Shanghai), 0.2M NaCl (Sinopharm, Shanghai), pH8.0). IB: washing buffer = 1g: 20ml, at room temperature, magnetically stirred for 30min, centrifuged at 8000g for 10min to remove the supernatant.

[0064] (2) Second wash

[0065] Inclusion bodies collected after the first wash were precipitated and washed with wash buffer (20 mM Tris-HCl, 2M urea, 5 mM EDTA, 0.2M NaCl, pH 8.0). IB: washing buffer = 1g: 20ml, at room temperature, magnetically stirred for 30min, centrifuged at 8000g for 10min to remove the supernatant.

[0066] (3) The third wash

[0067] Inclusion bodies collected after the third wash were precipitated ...

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Abstract

The invention provides fusion protein. The fusion protein is formed by connecting adjuvant protein with the end N or the end C of a tumor-associated antigen through a linker or a direct connecting mode. The adjuvant protein contains Haemophilus influenzae (Hi) protein D or a segment of the Haemophilus influenzae (Hi) protein D; the tumor-associated antigen contains HER2 protein with the amino acid sequence being SEQ ID NO:3 or a segment of the HER2 protein. The invention further provides a preparing method for the fusion protein and application thereof. The prepared recombined protein can restrain proliferation of HER2 positive breast cancer cells in a Blab / c mice body, or delay the increase speed of breast tumors.

Description

technical field [0001] The present invention relates to the fields of biology and medicine. Specifically, the present invention relates to a fusion protein capable of preparing a HER2 / neu antigen-positive tumor vaccine, specifically a fusion protein comprising Haemophilus influenzae (Haemophilus influenzae, Hi) protein D (ProteinD , PD) and HER2 / neu tumor antigen fusion protein. Background technique [0002] For the treatment of tumors, there are currently surgery, chemotherapy, radiation therapy and the like. Surgical therapy has no therapeutic significance for patients with multiple tissue metastases. Chemotherapy and radiation therapy can cause damage to normal cells due to their poor specificity. Therefore, there is a clinical need for a drug with strong specificity, which can specifically kill tumor cells without causing damage to normal cells. Tumor immunotherapy is to activate cytotoxic T lymphocytes (cytotoxic Tlymphocytes, CTL) against tumor antigens in vivo, and...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/285C07K14/47C12N15/70C12N15/80C12P21/02A61K38/16A61K39/00A61P35/00
Inventor 李树刚张伟辛渝但国平柴新娟程丹凝潘玉竹曹莉君于廷和
Owner 重庆科润生物医药研发有限公司
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