Construction of T7 phage display cDNA library from synovium of rheumatoid arthritis patients / 中国免疫学杂志
Chinese Journal of Immunology
;
(12)1999.
Article
in Chinese
| WPRIM
| ID: wpr-542200
ABSTRACT
Objective:
To construct and evaluate T7 phage display cDNA library from synovium of rheumatoid arthritis patients.Methods:
Total RNA was extracted from pooled RA synovium by Trizol reagents.Messenger RNA was isolated from total RNA by oligo (dT)-conjugated Oligotex particles,and then,the agarose gel electrophoresis showed the range of mRNA size.After mRNA was reverse-transcribed into double-stranded cDNA,end modification,adaptors ligation and EcoR Ⅰ and Hind Ⅲ digestion were performed.After eliminating excess adaptors and small fragments(less than 300 bp),the cDNA was ligated into T7Select 10-3 vector.The RA synovium phage display cDNA library was constructed by package reaction in vitro and plate proliferation.Plaque assay and PCR were used to evaluate the library.Results:
We showed that complexity of the library was about 2?107,and the phage titer of the amplified library was 8.9?10 10 pfu/ml.Insert ratio was proved to be 90% with the range of 300-2 000 bp inserts.Meanwhile,a target cDNA gene of BiPwas probed with PCR amplification.Conclusion:
The phage display cDNA library from synovium was construted with high quality and can be used as a valuable source in screening of RA self-antigens.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Immunology
Year:
1999
Type:
Article
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