CN1057128C - Sensory nerve antibody and preparing process - Google Patents
Sensory nerve antibody and preparing process Download PDFInfo
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- CN1057128C CN1057128C CN93111758A CN93111758A CN1057128C CN 1057128 C CN1057128 C CN 1057128C CN 93111758 A CN93111758 A CN 93111758A CN 93111758 A CN93111758 A CN 93111758A CN 1057128 C CN1057128 C CN 1057128C
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- sensory nerve
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Abstract
The present invention discloses a sensory nerve antibody and a preparing method thereof. The sensory nerve antibody can be specifically combined with a sensory nerve. The preparing method comprises the steps: the specific protein of the sensory nerve is taken as an antigen for immunizing a mouse, the cells of the immunized mouse are taken for conjugating and inoculating with myeloma cells, and then clones are cultivated and sieved so that hybridoma cell strains for secreting the sensory nerve antibody, and the like are obtained. The antibody is specifically combined with the sensory nerve, is conveniently for the separation of the sensory nerve and a motor nerve and has the advantages of simple preparing method, reliability and easy material acquirement.
Description
The method that the present invention relates to a kind of fermentation or use enzyme is a kind of peptide or protein and preparation thereof to synthesize desired compound or composition or separate optically-active structure body from racemic mixture, specifically a kind of monoclonal antibody and preparation thereof.
After the peripheral nerve injury, if the sensory tract and the mutual wrong of motor tract of two broken ends of fractured bone, the regenerated neural axis just can't arrive the identical nerve end-organ of function, and the skin of corresponding site and Muscle contraction function are all lost.Therefore, the function bundle of neural two broken ends of fractured bone accurately contraposition be one of neural essential condition of repairing the back functional rehabilitation.Around how could be accurately in the Shen Jing surgical repair, fast, identification function nerve tract easily, be the problem of concern very in orthopaedics and microsurgery, Neurological Surgery, plastic surgery and disabled and the rehabilitation medical.
Since using morphological approach difference nerve tract character the earliest from Australian Sunder Land in 1954, electrostimulation (Gruber1976), carbonic anhydride enzyme process (Piley 1984) and acetylcholinesterase methods such as (Cruber 1976, clock generation town 1987) has successively appearred.But these methods belong to non-specific explicit representation, influence its result's reliability, and long reaction time, therefore are very restricted in the prison bed is applied, and for these years, scholar both domestic and external is studying intensively always diligently, makes every effort to capture this difficult problem.
The object of the present invention is to provide a kind of can the combination with sensory nerve specifically, thereby can distinguish sensory nerve antibody of sensation, Motor nerve fibre and preparation method thereof.
Technical solution of the present invention is:
A kind of sensory nerve antibody can combine with sensory nerve specifically.
A kind of method for preparing sensory nerve antibody comprises the steps:
(a) get as antigenic mammals sensory nerve specific protein;
(b) use the antigen immune mouse, extract mouse cell then;
(c) will merge through the cell and the myeloma cell of mice immunized, and inoculate, cultivate the clone;
(d) detect antibody with the cellular immunization chemical method, only filter out Sensory neurone is played immunoreactive positive colony, more than three times, obtain the hybridoma cell strain of secretion sensory nerve antibody through the limiting dilution assay cloning again, CCTCCNO:C93004 secretes sensory nerve antibody.
The step of getting the mammals sensory nerve specific protein is: get the mammals nervous tissue, homogenate, centrifugal, extraction supernatant liquor are distinguished the distinctive zone of protein of sensory nerve with the isoelectric focusing sds gel electrophoresis, again with albumen recovery, purifying.The PH of isoelectrofocusing sds gel electrophoresis is controlled at 3-11.
Step with the antigen immune mouse is: inject sensory nerve protein 10 0 microgram with complete freund adjuvant balanced mix in mouse peritoneal, 2-4 is after week, again to sensory nerve protein 10 0 microgram of mouse peritoneal injection with incomplete freund adjuvant balanced mix, after 1-3 after week, sensory nerve protein 10 0 microgram, the splenocyte of extraction mouse after 2-4 days excessively reinject in mouse peritoneal.
To merge through the cell and the myeloma cell of mice immunized, and the step of inoculating, cultivating the clone is: will be through mice immunized splenocyte and myeloma cell, under the PEG effect, merge, with being inoculated in the Tissue Culture Plate that is covered with feeder cell after the HAT liquid dilution that contains calf serum, place 5% CO
237 ℃ of cultivations in the incubator, 2-4 changes HAT liquid after week continues to cultivate.
Sensory nerve antibody provided by the present invention can combine with sensory nerve specifically, so can (as the Coomassie brilliant blue mark) distinguish sensation and motorius easily after with the antibodies specific mark.The preparation method of sensory nerve antibody provided by the present invention, simple, reliable, material is easy to get.
The invention will be further described below in conjunction with embodiment:
Embodiment 1:
Get people's nervous tissue, homogenate, centrifugal, extraction supernatant liquor, sds gel electricity (PH3-11) is distinguished the distinctive zone of protein of sensory nerve and is reclaimed and the purifying sensory nerve specific protein, as antigen.
Select the BABL/C female mice (Shanghai Medical Univ provides) in eight ages in week for use.Sensory nerve protein 10 0 microgram of every interior injection of mouse peritoneal and complete freund adjuvant (Sigma) balanced mix, after three weeks, sensory nerve protein 10 0 microgram of every mouse peritoneal injection and incomplete freund adjuvant balanced mix, again after two weeks of interval, in mouse peritoneal, inject sensory nerve protein 10 0 microgram, got the splenocyte 1 * 10 of mouse in the 3rd day later
8With SP2/0 myeloma cell's (Chinese Academy of Sciences's Shanghai cell biological provides) 1 * 10
7PEG 50% (Shanghai washing composition two factories, divided amount 1000) effect fusion down.Be diluted to 100ml with the HAT liquid that contains 15% calf serum and contain 10
9Behind the individual cell, be inoculated in the 96 porocyte culture plates (Costar) that are covered with feeder cell (Turnover of Mouse Peritoneal Macrophages), every hole 0.2ml places 5% CO
2Incubator in (37 ℃) cultivate.Change HAT liquid after three weeks, at the bottom of clonal growth reaches the hole, 1/3 the time, detect antibody (detection reagent is provided by Shanghai Biological Products Inst., Ministry of Public Health), only filter out Sensory neurone is played immunoreactive positive colony with immunocytochemical method (ABC method).Clone more than three times through limiting dilution assay again, acquisition is secreted into this cell strain of hybridoma cell strain CCTCCNO:C93004 (Human SensoryNerve Antibody Cell) of sensory nerve antibody and promptly secretes people's sensory nerve antibody (HumanSensory Nerve Antibody writes a Chinese character in simplified form HSNA).This antibody can carry out specific antibody mark (or with other method mark) with the coomassie orchid.This antibody can combine with people's sensory nerve specifically.Can secrete the hybridoma cell strain of people's sensory nerve antibody, 96 of chromosome examination average out to can be inoculated in and produce ascites and knurl tubercle in the mouse body of the same race, this cell strain vitro culture is after 1 year, secretory antibody stably still, and preserve in this cell strain liquid nitrogen, recovery artifact activity is constant.
Embodiment 2:
Get the new zealand white rabbit nervous tissue of growing up, all the other are with embodiment 1, and the gained cell strain can be secreted the new zealand white rabbit sensory nerve antibody, and this antibody can combine with new zealand white rabbit is neural specifically.
Claims (5)
1, a kind of sensory nerve antibody is characterized in that: this antibody is by hybridoma cell strain CCTCCNO:C93004 excretory.
2, a kind of method for preparing sensory nerve antibody is characterized in that: comprise the steps:
(a) get as antigenic mammals sensory nerve specific protein;
(b) use the antigen immune mouse, extract mouse cell then;
(c) will merge through the cell and the myeloma cell of mice immunized, and inoculate, cultivate the clone;
(d) detect antibody with the cellular immunization chemical method, screening positive clone, only filter out Sensory neurone is played immunoreactive positive colony, more than three times, obtain the hybridoma cell strain CCTCCNO:C93004 of secretion sensory nerve antibody through the limiting dilution assay cloning again.
3, the method for preparing sensory nerve antibody according to claim 2, it is characterized in that, the step of getting the mammals sensory nerve specific protein is: get the mammals nervous tissue, homogenate, centrifugal, extraction supernatant liquor, distinguish the distinctive zone of protein of sensory nerve with the isoelectric focusing sds gel electrophoresis again, with albumen recovery, purifying.
4, the method for preparing sensory nerve antibody according to claim 3, it is characterized in that: the PH of isoelectric focusing sds gel electrophoresis is controlled at 3-11.
5, according to claim 2 or the 3 or 4 described methods that prepare sensory nerve antibody, it is characterized in that: the step with the antigen immune mouse is: inject sensory nerve protein 10 0 microgram with complete freund adjuvant balanced mix in mouse peritoneal, 2-4 is after week, again to sensory nerve protein 10 0 microgram of mouse peritoneal injection with incomplete freund adjuvant balanced mix, after 1-3 after week, sensory nerve protein 10 0 microgram, the splenocyte of extraction mouse after 2-4 days excessively reinject in mouse peritoneal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93111758A CN1057128C (en) | 1993-09-22 | 1993-09-22 | Sensory nerve antibody and preparing process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93111758A CN1057128C (en) | 1993-09-22 | 1993-09-22 | Sensory nerve antibody and preparing process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1084891A CN1084891A (en) | 1994-04-06 |
| CN1057128C true CN1057128C (en) | 2000-10-04 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93111758A Expired - Fee Related CN1057128C (en) | 1993-09-22 | 1993-09-22 | Sensory nerve antibody and preparing process |
Country Status (1)
| Country | Link |
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| CN (1) | CN1057128C (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1042734A (en) * | 1987-11-17 | 1990-06-06 | 美克德株式会社 | The monoclonal antibody of identification α 2 → 3 keys |
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1993
- 1993-09-22 CN CN93111758A patent/CN1057128C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1042734A (en) * | 1987-11-17 | 1990-06-06 | 美克德株式会社 | The monoclonal antibody of identification α 2 → 3 keys |
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| Publication number | Publication date |
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| CN1084891A (en) | 1994-04-06 |
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