[Cloning and secretory expression of islet neogenesis-associated protein in Pichia pastoris].
OBJECTIVE
To clone the recombinant human islet neogenesis-associated protein (rhINGAP) gene for its secretory expression in Pichia pastoris.


METHODS
INGAP gene was amplified with PCR and inserted between Xho I and EcoR I downstream sites of the alpha factor of the recombinant plasmid alpha/pUC18. The fusion gene of alpha factor and INGAP was subsequently inserted between BamH I and EcoR I sites of the plasmid pPIC9K of P. pastoris. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with Sal I digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay (ELISA).


CONCLUSIONS
The recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.
Sha
Jian-ping
JP
Department of Endocrinology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. shajianping99@sina.com
Xue
Yao-ming
YM
Chen
Xuan
X
Zeng
Zhan-jun
ZJ
Wei
Min
M
Luo
Xiang-rong
XR
He
Fei-ying
FY
Wang
Ling
L
Zhuo
Feng-ting
FT
D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D000073718:Pancreatitis-Associated Proteins; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D011994:Recombinant Proteins