[Expression of procalcitonin and characterization of antibodies against PCT].
OBJECTIVE
To construct the expression vectors of procalcitonin (PCT), prepare polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) against PCT and identify their specific biological activity.


METHODS
The recombinant expression plasmids of pGEX-4T-1-PCT and PET-32a-PCT were constructed using thyroid carcinoma cell line (TT cell) cDNA as template. The fusion protein of His-PCT was expressed in E.coli and used as immunogen. The specificity of antiserum against human PCT was characterized by ELISA, Western blot and indirect immunofluorescence. The mAbs against human PCT were identified by Western blot and indirect immunofluorescence.


RESULTS
The recombinant expression plasmids of pGEX-4T-1-PCT and PET-32a-PCT were constructed and the fusion protein of His-PCT was expressed and purified. The antiserum against human PCT was prepared and the titer detected by ELISA was 1:256 000. The pAb specifically recognized the recombinant human PCT. Eight hybridoma cell lines secreting specific mAbs against PCT were established. The mAbs recognized the recombinant human PCT and four of them recognized the native PCT of TT cytoplasm in immunofluorescent assay.


CONCLUSIONS
The successful preparation of polyclonal and monoclonal antibodies against human PCT is beneficial to further research into the pathological and physiological functions of PCT in severe bacterial infection and sepsis.
Liu
Qi
Q
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Institute of Radiation Medicine, Beijing 102206, China. qiliu_angel@yahoo.com.cn
Guo
Yan-Ru
YR
Liu
Ying
Y
Liu
Xiao-Lan
XL
Liu
Jing
J
Zhong
Fei
F
Gao
Jian-En
JE
Sun
Qi-Hong
QH
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D002116:Calcitonin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000235:genetics
D000906:Antibodies; D000911:Antibodies, Monoclonal; C000606589:CALCA protein, human; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide