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Osteoporosis vaccine built based on P-nitrobenzene alanine insertion method

A technology of nitrophenylalanine and nitrophenylalanine, which is applied in the direction of introducing foreign genetic material by using vectors, bone diseases, genetic engineering, etc., to achieve good prevention and treatment, break through immune tolerance, and overcome high cost problems Effect

Inactive Publication Date: 2014-08-06
陶惠人 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no vaccine that can overcome the body's immune tolerance to the autologous protein RANKL, thereby achieving a therapeutic effect on osteoporosis

Method used

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  • Osteoporosis vaccine built based on P-nitrobenzene alanine insertion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Construction and screening of p-nitrolated phenylalanine-substituted RANKL vaccine

[0017] (1) Construction of RANKL recombinant expression vector

[0018] Purchase the RANKL cDNA clone, amplify the coding sequence of the extracellular segment of RANKL by PCR, digest the PCR product with restriction enzymes, and connect it into a vector (such as pET28a, etc.) that has also undergone the above-mentioned double enzyme digestion to construct the corresponding recombinant expression vector (such as pET28a-RANKL recombinant expression vector). The correctness of the sequence was confirmed by bidirectional sequencing.

[0019] (2) Site-directed mutation of the RANKL gene (take the mutation of the site encoding tyrosine as an example)

[0020] ①Three-dimensional structure analysis of RANKL protein to determine the tyrosine residues for mutation / substitution. The crystal structure data of RANKL was retrieved from the protein database, and the protein structure wa...

Embodiment 2

[0028] Example 2: Therapeutic Effect of p-Nitrated Phenylalanine-RANKL Recombinant Vaccine on Osteoporosis Mice

[0029] 1) Preparation of mouse osteoporosis model According to related literature, we used female C57BL / 6 mice to undergo ovariectomy to simulate a postmenopausal osteoporosis model.

[0030] 2) Observation of animal immunity and biological effects: 48 female C57BL / 6 mice aged 12 weeks were selected and randomly divided into four groups (12 mice in each group).

[0031] One group: antigen immunization + ovariectomy

[0032] The second group: antigen immunization + sham operation

[0033] Three groups: PBS injection + ovariectomy

[0034] Four groups: PBS injection + sham operation

[0035] The experimental animals were kept in separate cages at 25±2°C and well ventilated, and paired with food and water. Groups 1 and 2 were injected subcutaneously with 10 μg / mouse of p-nitrophenylalanine-RANKL recombinant vaccine near the lymph nodes on days 0, 14, 21, 42 and 63...

Embodiment 3

[0045] Embodiment 3: Observation of the safety of RANKL recombinant vaccine

[0046] Mainly observe the effect of p-nitrolated phenylalanine-RANKL recombinant vaccine on the immune system and thermoregulation function

[0047] 1) The composition of whole blood cells: the number / ratio of red blood cells, white blood cells, lymphocytes and platelets is detected by a blood cell counter; the composition of lymphocytes (B lymphocytes, T lymphocytes (CD4+ and CD8+) is changed by flow cytometry ).

[0048] 2) Lymph node and spleen tissue sections: The mesenteric lymph nodes and spleens of 3 mice were sampled from each group of mice, fixed in 10% formalin, coated with paraffin, and cut into 5 μm tissue sections. Deparaffinize, perform HE staining and immunohistochemical staining with anti-CD3 antibody respectively, and observe whether there are abnormal changes in the main lymphoid tissue structure of mice immunized with p-nitrophenylalanine-RANKL recombinant vaccine.

[0049] 3) Im...

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Abstract

The invention discloses an osteoporosis vaccine built based on a P-nitrobenzene alanine insertion method. The osteoporosis vaccine is characterized by being prepared through the following steps: 1), the construction of RANKL recombinant expression vector; 2), the fixed point insertion / replacement of the coded sequence of an unnatural amino acid, namely p-nitrobenzene alanine; 3), the recombination expression and purification of p-nitrobenzene alanine-RANKL albumen; 4), the animal immunization and screening; 5), the screening based on serum neutralization detection and osteoclast formation analysis; 6), the animal experiment, and the determination of required vaccine. According to the invention, the method can effectively break through immune tolerance of the human body to autologous albumen, and induces the human body to generate special neutralizing antibody for RANKL through an active immune means, so as to achieve the purpose of preventing osteoporosis well; besides, the problem of high cost for industrially producing antibodies is solved or avoided.

Description

technical field [0001] The invention relates to a medicine for treating osteoporosis, in particular to a vaccine for producing antibodies against osteoclast differentiation factor receptor ligand (RANKL) constructed based on the P-nitrophenylalanine insertion method. It also relates to a preparation method of the medicine. Background technique [0002] Osteoporosis is mainly a systemic bone metabolic disorder characterized by a reduction in bone mass and destruction of bone microstructure, which reduces the bearing capacity and strength of the bone and is prone to brittle fractures. It is also a variety of bone destructive diseases. Shared pathological damage. The incidence of osteoporosis is high, and there is an increasing trend year by year. Osteoporotic fractures seriously affect the quality of life of patients, threaten their life safety, and also bring a heavy economic burden. The current drugs for the treatment of osteoporosis include calcium, vitamin D3, bisphosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00C12N15/12C12N15/63C07K14/47A61P19/10
Inventor 陶惠人李锋
Owner 陶惠人
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