Cloning,Expression Of Apr-2 And Preparation Of Polyclonal Antiserum

Somatic growth and development are homeostatic regulatory processes between cell proliferation and cell death. The latter case can be encountered in normal cells and is thus referred to as programmed cell death (PCD) or apoptosis. Apoptosis has diverse yet important roles under both physiological and pathological situations, which is governed by a plethora of relevant genes and gene products that either dictate cell death or prolong cell survival. It is believed that apoptosis is involved in proapoptotic genes, antiapoptotic genes and those genes expressed during apoptosis. Several studies have shown that apoptosis is closely related to hematopoietic system, malignant disease and infectious disease and etc. Several reasearchers have attempted to induce apoptosis in cancer cells by triggering the core components of the cell-death machinery such as the BCL2 family of proteins, the intracellular antiapoptotic proteins (lAPs), tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and the caspases. Inducers of apoptosis have been used in cancer therapy, and some of them have shown promising results. Better knowledge of cellular mechanisms that regulate apoptosis will provide a strong rationale for the individualization of chemotherapy regimens and contribute to develop more selective apoptosis inducers to minimizeside-effects and maximize efficacy. Thus every novel gene related to apoptosis will be accompanied by further understanding of mechanism of apoptosis. Zhu Feng and others found a series of genes in HL-60's apoptosis model induced by ATRA using improved substractive hybirdization. They thought that these genes might be related to apoptosis ,and named apr-l~apr5.The full length of apr-2 is 791bp, and it locates on chromosome 17. The length of apr-2 cDNA coding region is 339bp and codes 111 amino acids. Little is known about apr-2 so far, so it is necessary to clone this gene, which is the basis for further study. Three experiments were included as follows:1. Established the apoptotic model of HL-60, and total RNA was isolated from the cells and mRNA was reversely transcribed into cDNA. Then PCR was used to amplify the apr-2 coding region. The PCR product was cloned into PGEM-T Easy vector and sequenced.2. Target gene was subcloned into expression vector PGEX-4T-2 and induced with IPTG. Analyzed the distribution of expressed products by Band density scanning of stained gel. Preliminarily purified fusion protein of GST-APR-2 by electric elution.3. The purified fusion protein was mixed with Freund's complete or incomplete adjuvant, and then used to immunize rabbits. Detected liter of antiserum by means of ELIS A and Western-blot.4. Extracted the total protein from HL-60 cells, which had been treated with ATRA for 12 h. Detected the existence of APR-2 through the method of Western-blot.RESULTS: Coding region of apr-2 cDNA was synthesized by RT-PCR. The amplified coding region of apr-2 cDNA was identified by electrophoresis on agarose gels, and obtained approximately 339bp band as expected, apr-2 was cloned into PGEM-T Easy vector and the sequence was confirmed. SDS-PAGE showed that the fusion protein was expressed as inclusion bodies in E.coli. Band density scanning of stained gel was performed to estimate the percentage of the recombinant protein in the total bacteria protein , which was up to 30%. The titer of antiserum was 1:5000. Western Blot analysis showedthe antiserum could combine with prokaryotic expressed fusion protein. The expression of APR-2 protein was confirmed by SDS-PAGE and Western Blot analysis in cell split products after 12h induced by ATRA with the antiserum we produced.CONCLUSION: apr-2 was successfully cloned and obtained expression product of fusion protein. Obtained the APR-2 antiserum from rabbits. Preliminarily confirmed the APR-2 expression in the apoptotic HL-60 cell. Laid the foundation for further studies of apr-2's structure-function and the mechanism of apoptosis.