[Cloning of mouse TLR-2 gene and its expression in Pichia pastroris].
OBJECTIVE
To clone the mouse TLR-2 gene and to express it in Pichia pastroris.


METHODS
Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot.


RESULTS
The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody.


CONCLUSIONS
The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.
Zhao
Wen-zhong
WZ
Department of Immunology, The First Military Medical University, Guangzhou 510515, China. zhaowz@fimmu.edu.cn
Jiang
Hai-yan
HY
Liu
Yan-jun
YJ
Zhu
Ping
P
Fu
Ning
N
D000818:Animals; D003001:Cloning, Molecular; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D051195:Toll-Like Receptor 2; D014170:Transformation, Genetic
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2