[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].
OBJECTIVE
To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain.


METHODS
The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA.


RESULTS
The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically.


CONCLUSIONS
The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.
Fan
Ai-lin
AL
Department of Clinical Laboratory, Fourth Military Medical University, Xi'an 710032, China. fanailin@fmmu.edu.cn
Shi
Chang-hong
CH
Su
Ming-quan
MQ
Ma
Jing
J
Bai
Yin-lan
YL
Cheng
Xiao-dong
XD
Xu
Zhi-kai
ZK
Hao
Xiao-ke
XK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D016207:Cytokines / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016982:Micrococcus luteus / Q000737:chemistry*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D016207:Cytokines; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C113704:resuscitation-promoting factor, bacteria