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Initial Study of the Function NBEAL1 Gene and Protein

Author: BaoChenChen
Tutor: CuiDaXiang
School: Shanghai Jiaotong University
Course: Biochemistry and Molecular Biology
Keywords: NBEAL1 (Neurobeachin Like 1) Clone Expression Purification Antibody preparation Glioma Pathological grade
CLC: R739.4
Type: Master's thesis
Year: 2010
Downloads: 14
Quote: 0
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Abstract


Objective: To construct a new gene Neurobeachin Like 1 (NBEAL1) expression vector , expression and purification of recombinant protein and identification of its biological activity , preparation of monoclonal antibodies and detection NBEAL1 protein expression in the different degree of malignancy of brain glioma . : By first extracting the total RNA of normal brain tissue , reverse transcription cDNA, screening the appropriate fragment build pGEX-KG/NBEAL1 expression vector , transformed into E. coli BL21 (DE3 ) , IPTG -induced expression of GST protein affinity purification Western Blotting were identified purity and specificity of the purified product , the preparation of monoclonal antibodies, and using the antibodies prepared to study the role of the protein in the variation of the degree of malignancy of the tumor . : NBEAL1 gene fragment was cloned into the pGEX-KG expression vector sequencing confirmed the correct sequence of the cloned fragment , NBEAL1 fusion protein expression in inclusion bodies , expression accounts for more than 30% of the total bacterial proteins , the purified recombinant protein purity of 95% or more. The Western Blotting prove purified protein containing the GST tag NBEAL1 peptides . 2 monoclonal antibody was successfully prepared . Low expression of the protein in the normal brain tissue , upregulated in glioma tissues of the low -level , and the higher the degree of malignancy , the lower the degree of expression of NBEAL1 negative correlation . Conclusion : NBEAL1 gene fragments were successfully cloned , expression and purification ; monoclonal antibody was successfully prepared ; glioma expression levels of malignant glioma degree of correlation . This study has laid a foundation for further elucidate NBEAL1 gene 's role in the the gliomas development process .

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