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Abstrak :
Keamanan dari vaksin terapetik untuk kanker serviks yang didasarkan pada antigen HPV E6 perlu dipastikan dengan uji interaksi antara vaksin dan protein p53. Tujuan dari penelitian ini adalah untuk mendapatkan plasmid rekombinan untuk ekspresi protein rekombinan p53 yang akan digunakan dalam uji interaksi. Enzim RevertAid dan primer random hexamer digunakan untuk mendapatkan cDNA dari sel HeLa yang akan digunakan sebagai cetakan PCR dengan menggunakan enzim Platinum Taq DNA Polymerase High Fidelity dan primer p53 spesifik. Produk PCR yang dihasilkan sebesar 1204 bp yang mengandung gen p53 dengan adanya penambahan situs restriksi endonuklease SalI dan PstI. Setelah pemotongan dengan enzim SalI dan PstI, produk PCR diligasi dengan plasmid pQE-80L yang juga sudah dipotong dengan enzim yang sama. Campuran ligasi digunakan untuk transformasi ke dalam Escherichia coli TOP10. Keberadaan fragmen DNA p53 yang berhasil dimasukkan ke dalam plasmid rekombinan yang sudah berada di dalam transforman diverifikasi menggunakan PCR, pemotongan DNA rekombinan, dan sekuensing. Hasil penelitian menunjukkan bahwa gen p53 manusia berhasil diklon ke pQE-80L dengan adanya mutasi pada urutan basa nukleotida ke-386.

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In order to confirm the safety of a therapeutic vaccine for cervical cancer that is based on HPV E6 antigen, an interaction assay between the vaccine and the p53 protein is required. The aim of this study is to obtain a recombinant plasmid for expression of p53 recombinant protein that will be used in the interaction assay. The RevertAid enzyme and random hexamer primer was employed to obtain cDNA from HeLa cell that will be used as template in PCR using the Platinum Taq DNA Polymerase High Fidelity enzyme and p53 specific primer set, to generate a 1204 bp PCR product containing the p53 gene, with flanking SalI and PstI endonuclease restriction sites. Following restriction with SalI and PstI enzyme, the PCR product was ligated with the plasmid pQE 80L that has been linearized by restriction with the same enzymes. The ligation mixture was used for transformation of Escherichia coli TOP10. The presence of the inserted p53 DNA fragment in the recombinant plasmid harboured by the transformants was verified using PCR, digestion of recombinant plasmids, and sequencing. The results showed that the human p53 gene was successfully cloned into pQE 80L with a mutation at position 386 of the p53 nucleotide base sequence.

Abstrak :
Selama ini pengobatan kanker serviks hanya menggunakan vaksin profilaktik yang bersifat preventif. Pengembangan vaksin terapeutik yang bersifat kuratif perlu dilakukan untuk penderita yang berada dalam tahap terinfeksi HPV-16 pra-kanker dan kanker. Akan tetapi, efektifitas dan keamanan kandidat vaksin terapeutik perlu diuji terlebih dahulu dengan uji interaksi onkoprotein E6 dengan protein penekan tumor p53. Oleh karena itu, tujuan penelitian ini adalah melakukan ekspresi protein p53. Protein p53 merupakan salah satu komponen sistem pendeteksi interaksi antigen E6 dengan p53. Ekspresi p53 menggunakan sel E. coli transforman dilakukan dengan pemberian induksi IPTG 1 mM selama 4 jam. Plasmid rekombinan pQE-80L_p53 mampu mengekspresikan protein p53 di dalam sel E. coli BL21 cp dengan berat molekul 54 KDa. Hasil western blotting menunjukkan sebuah pita berukuran 54 KDa yang sesuai dengan berat protein p53. ...... Over the last few years, cervix medication depends only on prophylactic vaccination. The development of therapeutic vaccination needs to be improved in order to treat HPV 16 infected patients. However, vaccines safety needs to be tested by examining interaction between E6 oncoprotein and p53 tumour suppressor protein. Therefore, p53 protein needs to be expressed as one of the system components. pQE 80L recombinant plasmid is capable to express p53 6xhis tagged using E. coli BL21 Codon Plus as a cell host. Western blot result showed that 4 hours of 1 mM IPTG induction produced a single band with the size of 54 kDa.

Abstrak :
Bacterial lipopolysaccharide (LPS) is impacted in the etiology of inflammatory periodontal disease. Aside from immunopathologic reactions which may be involved in the pathogenesis of the disease, the possibility exist that direct cytotoxic effect on cultured human gingival fibroblasts may be equally destructive. The expression of P53 protein can be one of markers to examine the state of impaired DNA. The purpose of this study was to investigate the effect of LPS toward expression of P53 protein on cultured human gingival fibroblasts. Cultured human gingival fibroblasts were exposed to LPS in concentrations of 50 and 200 ug/ml and untreated medium for a period of 24 and 48 hours. Cells were harvested and prepared for immunohistochemical evaluation. After exposure for 24 and 48 hours, the fraction of P53-positive cells was 81.7% in case of 50 ug/ml LPS, and 88.8% in case of 200 ug/ml LPS. After exposure for 48 hours, the fraction of P53-positive cells was 32.2% in case of 50 ug/ml LPS, and 21.1% in case of 200 ug/ml LPS. None of untreated group showed p53-positive cells. Up-regulation of p53 protein during the initial logarithmic phase of growth may be a consequence of on-going DNA damage.