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[Preparation of anti-LRRC4 polyclonal antibody and its application in constructing expression profile of human
Dan Li1, Ming-hua Wu, Qiong Chen
1Cancer Research Institute, Central South University, Changsha 410078, China.
Objective:
To prepare anti-LRRC4 polyclonal antibody and analyze the correlation between the expression of LRRC4 and pathological grades of gliomas in rabbits.
Methods:
Appropriate protein sequence with good hydrophilicity and antigenicity was chosen by analyzing with DS Gene 1.1 software. The corresponding nucleic acid sequence amplified by PCR was used to construct a recombinant pGEX-4T-2/276 bp. E.coli JM109 transformed with the recombinant was induced by IPTG to express GST-fusion protein, and the fusion protein expressed as insoluble inclusion bodies. Then the purified inclusion body was used to immunize rabbits. Once the titer of antiserum reached 1:10(8) by indirect ELISA, the serum was collected and purified. The expression-profile of LRRC4 in embryonic tissues and gliomas with various pathological grades were obtained by western blot and immunohistochemistry with the anti-LRRC4 polyclonal antibody.
Results:
The highly specific anti-LRRC4 polyclonal antibody whose titer reached 1:10(8) was prepared. The relatively specific expression of LRRC4 was detected in the normal brain, but reduced expression or loss of expression in gliomas was also noticed by immunohistochemistry, and there was a correlation between the expression level of lrrc4 and the pathological grade of gliomas.
Conclusion:
The anti-LRRC4 polyclonal antibody with high titer and specificity has been obtained. A correlation between the expression level of LRRC4 and the pathological grade of gliomas is detected, which lays the foundation for advanced research of LRRC4.
