A method for screening autoantigens
A self-antigen and antigen technology, applied in the field of cell biology and protein, can solve the problems of limited ability of membrane protein, heavy workload, large amount of control serum, etc.
Active Publication Date: 2016-08-03
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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Problems solved by technology
[0005] Although there have been many reports on the use of SERPA technology in the screening of autoantigens [10-21] , but its main disadvantages are some defects of 2-DE itself, such as the limited ability to separate extremely acidic, extremely basic proteins and poorly water-soluble membrane proteins, and the limitation of detection sensitivity can only identify relatively high-abundance antigens. In addition, 2-DE It is a labor-intensive experiment, the more serum tested, the greater the workload
At the same time, the amount of patient or control serum required is relatively large
The second broad category of approaches is the evaluation of existing antigens, rather than the discovery of novel autoantigens
Method used
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Embodiment 1
[0031] Example 1: Identification of tumor-associated antigens in esophageal squamous cell carcinoma
[0032] Cell culture: Esophageal squamous carcinoma cell lines EC0156 and KYSE410 (EC0156 cell lines are from the Institute of Cancer, Chinese Academy of Medical Sciences, references: WangQ, XuY, ZhaoX, ChangY, LiuY, JiangL, SharmaJ, SeoDK, YanH.Afacileone-step insitufunctionalizationofquantumdotswithpreservedphotoluminescenceforbioconjugation. JAmChemSoc2007; 129: 6380-6381; KYSE410 cell line was a gift from Dr. Toyoshi Shimada, Hyogo Medical College, Japan)
[0033] 1) EC0156 and KYSE410 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and RPMI1640 medium, respectively, and the medium contained 10% inactivated fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin in 5% CO 2 cultured in a 37°C incubator (NAPCO, Winchester, VA).
[0034] 2) Apply the subcellular protein extraction kit "ProteoExtract TM Kits" (Cat.No.539790, MERCK Company) divided ...
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The invention relates to the field of molecular biology, in particular to an improved method for screening self-antigens. The present invention provides the following methods: dividing cell protein into subcellular components; performing preliminary screening by an improved one-dimensional Western blot method; and performing definitive screening by two-dimensional Western blot. It solves the problems of low-abundance component loss and low screening efficiency in conventional screening methods. The method can comprehensively and efficiently screen out autoantigens from proteins extracted from cells, and is useful for autoimmune diseases, tumors, and other diseases that can produce autoantibodies. Drug preparation for disease provides useful targets.
Description
technical field [0001] The invention relates to the fields of cell biology and protein, in particular to a method for rapidly screening self-antigens. The method of the invention can effectively screen self-antigens from protein extracted from cells within several months, and is suitable for providing useful targets for drug preparation of various diseases capable of producing autoantibodies, such as autoimmune diseases and tumors. Background technique [0002] When factors such as biology, physics, chemistry and drugs act on the body, self-antigens can be changed, antigens released from immune isolation sites, molecular mimicry effects can be generated, epitope expansion and immune neglect can be broken, and these changes can cause autoimmune diseases. In the 1970s, it was discovered that autoantibodies and (or) autoreactive T lymphocytes could be detected in tumor patients, confirming the existence of tumor antigens (a type of autoantigens) [1] . At present, it is believ...
Claims
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IPC IPC(8): G01N33/53G01N27/64
Inventor 赵晓航高红军乔媛媛
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI