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A kind of nano antibody against rabies virus g protein and its application

A nanobody, rabies virus technology, applied in antiviral immunoglobulins, antiviral agents, viral antigen components, etc., can solve the problems of lack of CH1 in heavy chain, lack of light chain binding ability, inability to express heavy chain CH1 domain, etc.

Active Publication Date: 2021-05-04
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This heavy chain-only antibody may be due to mutations and deletions at the genomic level that lead to the inability to express the CH1 domain of the heavy chain, so that the expressed heavy chain lacks CH1, thereby lacking the ability to bind to the light chain, thus forming a heavy chain. chain dimer

Method used

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  • A kind of nano antibody against rabies virus g protein and its application
  • A kind of nano antibody against rabies virus g protein and its application
  • A kind of nano antibody against rabies virus g protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1. Preparation of rabies virus G protein

[0044] 1.1 Transfer vector construction

[0045] The rabies virus G protein extramembrane region (AAV85905.1) plasmid PUC57-RVG (synthesized by Jinweizhi Company, containing a His tag at the C-terminus) and the carrier PFastBac HTB (purchased from Biofeng Company) were double-introduced by BamⅠ and PstⅠ simultaneously. Digest with Dicer, recover the G protein extramembrane region gene (1.4kb) and the linearized target vector fragment (4.8kb), use T 4 Ligase ligation, named PFastBac HTB-RVG, transformed into DH5α host bacteria, screened for double resistance to gentamicin and ampicillin, selected positive colonies, amplified and extracted plasmids, and identified by double enzyme digestion with BamⅠ and PstⅠ and sequenced ( figure 1 : M is Trans 2K DNAmarker; 1 is the product of PFastBac HTB-RVG digested by BamI and PstI).

[0046] 1.2 Construction of expression plasmid

[0047] After overnight amplification and e...

Embodiment 2

[0053] Example 2. Construction and screening of anti-rabies virus G protein nanobody phage display library

[0054] 2.1 Immunity of alpacas

[0055] Select a healthy adult alpaca, mix the purified rabies virus G protein with Freund's adjuvant at a ratio of 1:1, and immunize the alpaca with 6-7μg / kg subcutaneous injection at multiple points on the back. times, the immunization interval was 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0056] 2.2. Isolation of camel-derived lymphocytes

[0057] Lymphocytes were separated from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, and each 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store the rest at -80°C.

[0058] 2.3 Total RNA extraction

[0059] Total RNA was extracted according to routine procedures in the technical field, and the concentrati...

Embodiment 3

[0092] Example 3. Affinity activity determination of anti-rabies virus G protein nanobody and antigen

[0093] 3.1 Chip antigen coupling: Prepare the antigen with different pH sodium acetate buffer (pH5.5, pH5.0, pH4.5, pH4.0) to make 20μg / mL working solution, and prepare 50mM NaOH regeneration solution at the same time, The template method in the instrument of Biacore T100 protein interaction analysis system was used to analyze the electrostatic binding between the antigens at different pH conditions and the surface of the chip (GE Company). Use a neutral pH system and adjust the antigen concentration as required for coupling conditions. The chip was coupled according to the template method included in the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical coupling amount. The coupling process takes about 60 min.

[0094]3.2 Exploration of analyte concentration setting condi...

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Abstract

The invention discloses a nanobody against rabies virus G protein. The nanobody has three unique complementarity determining regions CDR1, CDR2, and CDR3. The invention also provides the preparation of the nanobody for the prevention and / or treatment of rabies. application in medicine. The nanobody provided by the present invention has highly specific recognition and binding ability to rabies virus G protein, and the affinity constant can reach up to 1.36E-10, and can competitively inhibit G protein and newborn SD by binding specifically to G protein The combination of rat brain nerve cell receptors proves that it can play an effective immune protection role in the prevention and / or treatment of rabies virus infection, and shows that it has a good application prospect in the preparation of rabies prevention and / or treatment drugs.

Description

technical field [0001] The invention discloses an antibody, more specifically, the invention discloses a nanobody. Background technique [0002] Rabies is a zoonotic global infectious disease caused by rabies virus. Once people and animals get sick, almost 100% of them die. WHO recommends that for rabies exposure, especially severe exposure, rabies vaccine and anti-rabies antibody should be injected throughout the course. Antiserum plays a vital role in the prevention of rabies. Due to the large side effects of equine immune serum (ERIG), the source of human rabies immunoglobulin (HRIG) is limited, and there are potential pathogen contamination and batch-to-batch differences, etc. Disadvantages, therefore, there is an urgent need to develop human anti-rabies monoclonal antibodies or humanized genetically engineered antibodies for therapeutic use. At the same time, whether it is the titer detection during the production process of rabies vaccine or the detection of anti-rab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21A61K39/12A61P31/14C12R1/19
CPCA61K2039/505A61P31/14C07K16/10C07K2317/22C07K2317/565C07K2317/567C07K2317/569C07K2317/92
Inventor 宋海鹏刘原源黄琪陈晓恒曹慧王欢
Owner 深圳市国创纳米抗体技术有限公司
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