Institut Penyelidikan Perubatan Molekul - Tesis

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Antiaging Activity Of Polyphenol Rich Calophyllum Inophyllum Fruit Extract In Aging Mutants Of S. Cerevisiae
(2022-12)
S. Venugopal, Kavilasha
Calophyllum inophyllum L. is an important medicinal plant with many ethnomedicinal values and exhibited significant anti-oxidant activity. Hence, this study was conducted to determine the in vitro anti-aging property of C. inophyllum fruit extract (CIFE) against Saccharomyces cerevisiae BY611 Strain with the molecular mechanism of the anti-aging process. The anti-aging effect of CIFE against the yeast was evaluated through chronological lifespan assay, anti-oxidative stress assays, real-time quantitative polymerase chain reaction (RT-PCR) to study the regulation of superoxide dismutase (sod) and sirtuin 1 (sirt1) genes and superoxide dismutase enzyme (SOD) and sirtuin 1(SIRT1) protein activity assays were conducted at 1 mg/mL of CIFE. After administrating CIFE, the lifespan of the yeast was significantly prolonged in comparison with the untreated group (P<0.05) and also increases the yeast cell viability under oxidative stress by 4 mM H₂O₂ by spot assay. The Phloxine B stain method also further proves yeast cells' viability increased under oxidative stress by CIFE treatment. The reactive oxygen species (ROS) assay revealed that CIFE notably reduces the oxidative stress in treated yeast cells (P<0.05) comparing to untreated yeast cells.
Assessment Of Antitumor Activity Of Standardized Polyalthia Longifolia Leaf Extract In Hela Cell Tumor Xenograft Mouse Model
(2022-08)
Braganza, Cilwyn Shalitha
P. longifolia is a traditional medicinal plant with a rich source of biologically active phytochemicals. Investigations on methanolic leaf extract were reported to exert potent in vitro anticancer effects on HeLa human cervical cancer cells. Current treatment strategies for cervical cancer are facing challenges such as increased drug toxicity, chemoresistance, and limited treatment options, thus making it imperative for the discovery of safe and effective green anticancer agents. Though P. longifolia is a promising anticancer plant, the in vitro findings by themselves are insufficient to translate to human use and therefore require in vivo preclinical testing. Currently, there are no reports on further investigation of P. longifolia leaf extracts on in vivo animal tumor models, which limits the clinical utility of this plant. Hence this study was conducted to evaluate the in vivo antitumor effects of P. longifolia methanolic leaf extract on HeLa cells xenografted tumors developed in athymic nude mice.
Bzd9l1: Elucidation Of Its Anti-angiogenic Potential In In-vitro And In-vivo Colorectal Cancer Models
(2023-09)
Subramaniam, Ayappa V.
Colorectal cancer (CRC) is the third most common cancer globally. CRC depends largely on angiogenesis for growth and metastasis. Much effort has been made to selectively target the angiogenic pathways to restrain tumour growth. However, some CRC patients become resilient to these anti-angiogenic drugs and standard therapies. The class III histone deacetylase family of sirtuins (SIRTs) has been closely linked to cancer progression but less is known about its activity in regulating tumour angiogenesis. BZD9L1 is a novel sirtuin inhibitor with demonstrated anti-cancer activities. This study aimed to investigate the anti-angiogenic potential of BZD9L1 on EA.hy926 endothelial cells (EC) in vitro and HCT116 tumour xenograft nude mice. The in vitro experiments comprised of cell viability assay, scratch wound assay, tube formation assay, spheroid sprouting assay, western blotting, angiogenesis array, cell cycle and apoptosis analysis via flow cytometry and finally, indirect co-culture model. Nude mice tumour xenograft model was used for the in vivo study, where hematoxylin and eosin staining was done to study the percentage of necrosis in the tumour section and immunohistochemistry was conducted to investigate the protein expression of Ki67 and CD34. BZD9L1 was shown to reduce cell viability, cell migration, tube formation, and spheroid sprouting of EC. BZD9L1 at 10μM was also shown to inhibit SIRT2 and SIRT3 protein in EA.hy926 cells. Angiogenesis array results revealed that the compound reduces the cytokine concentration of Angiogenin, bFGF, PDGF-BB, and PIGF significantly (P * < 0.05) compared to the control group
Characterization Of Astaxanthinrich Xanthophyllomyces Dendrorhous Extract From A Hyperproducing Mutant And Its Effects On Breast Cancer Cells
(2022-12)
Khaw, Shin Yuan
Astaxanthin is a carotenoid with multiple health benefits including antioxidant and anticancer properties. An astaxanthin-hyperproducing X. dendrorhous mutant was generated due to the growing preference for natural pigment. This work aimed to study the astaxanthin-rich X. dendrorhous extracts and its effect on MCF-7 and MDA-MB- 231 cells. Spectrometric, TLC and HPLC analysis evidenced that mutant M34 produced astaxanthin in higher quantity and purity than the wild type. Mutant astaxanthin extract showed a higher scavenging activity compared to wild type astaxanthin extract in DPPH assay. MTT assays proved that wild type and M34 astaxanthin extracts were not toxic towards the non-cancer origin MCF-10A cells. Wild type and mutant astaxanthin extracts exhibit growth-inhibitory effect in dosedependent manner in MCF-7 and MDA-MB-231 cells. Mutant astaxanthin extract showed a lower IC50 than wild type extract and was more effective in inhibiting both MCF-7 and MDA-MB-231 cells. The IC50 values of wild type and mutant astaxanthin extracts did not exceed the required upper limit of 30 μg/ml.
Chemopreventive Effects And Proteomic Analysis Of Myo Inositol Treatment On Human Prostate Cancer Cell Line (Du-145)
(2022-02)
Islam, Mohammad Jahidul
Prostate cancer remains the second most frequent diagnosed cancer in men and is the third leading cause of cancer related death, despite many available treatment options. The inefficiency of existing therapies and their adverse effects have led scientists to seek new treatments for this disease. The identification and development of new anti-proliferative agents for prostate cancer is a major innovation in treating this deadly disease. Although the synthetic compound Myo-inositol is widely used in cancer research, the actual anti-cancer function of these chemicals is still not well understood. This study was therefore intended to unveil the chemopreventive effects of Myo-inositol on prostate cancer cells (DU-145). The growth inhibitory effect and the IC50 value were demonstrated by Myo-inositol at 0.06 mg/ml (**p<0.05). It is suspected that cell deaths are related to apoptosis induction and cell-cycle arrest. Treatment with Myo-inositol has been further identified by induction of early and late apoptosis (***p<0.01). Apoptosis can also be detected using DNA fragmentation and Hoechst 33342 fluorescent dye stain analysis. Myo-inositol caused an alteration to the cell cycle regulation on DU-145 cell line at G0/G1 and S phase, respectively (***p<0.01). Protein identification results via 2D electrophoresis and LC-MS/MS analysis showed 19 and 23 proteins identified in control and Myo-inositol treated samples, respectively.
Deciphering S-Phase Protein Kinase 2 (Skp2) Mediated Regulation Of Telomerase Reverse Transcriptase (Tert) In Kasumi-1, T (8;21) Acute Myeloid Leukemia (Aml) Cell Line
(2023-06)
Rosli, Nur Aliaa Arina
Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is characterized by immature myeloid cell proliferation. S-Phase Protein Kinase 2 (SKP2) is a cell cycle regulator and shown to be overexpressed in AML patients. SKP2 potentiates to cause cell proliferation and division. Studies shown prolonged SKP2 knockdown suppress telomerase reverse transcriptase (TERT) expression in t(8;21) AML cells in vitro. Nevertheless, the molecular mechanism of TERT regulation by SKP2 remain unclear. Therefore, the aim of this study was to investigate the TERT mechanism by SKP2 in AML.SKP2 was suppressed in Kasumi-1 and THP-1 via siRNA mediated gene knockdown. In this study, TERT expression reduced at gene and protein level after SKP2 suppression in non t(8;21) AML cells. Accordingly, telomerase activity was also reduced in non t(8;21) AML cells. Result obtained show that c-Myc and FOXO3 did not play a direct role in SKP2 mediated TERT regulation in t(8;21) and non-t(8;21) AML cells at gene level. However, different pattern in c-Myc protein expression was observed in non t(8;21) AML cells. Another observation were made in t(8;21) AML cells after AML1/ETO knockdown where c-Myc was up-regulated at gene and protein level. Due to increase in c-Myc expression levels, chromatin immunoprecipitation (ChIP) was carried out and increased observed binding of c-Myc to the TERT promoter was observed after AML1/ETO down-regulation. However, c-Myc binding to the TERT promoter failed to induce TERT transcription in t(8;21) AML cells.Other proteins related to TERT mechanism xxii observed were Rb, E2F1 and CDKN1B. Hypophosphorylation of Rb was observed up-regulated in non t(8;21) AML cells after SKP2 knockdown yet no significant difference in E2F1 protein expression was observed. Accumulation of CDKN1B was markedly related with suppression of SKP2 in this studies.
Development Of A Flexible Stable Mammalian Antibody Expression Pipeline
(2023-02)
Jacqueline Mark Kar Kei
Mammalian antibodies are promising tools for biopharmaceutical use because of their high specificity and low toxicity. These proteins are favorable for both therapeutic and diagnostic purposes and its ever-growing demand calls for high productivity cell lines. Mammalian cell lines can express antibodies transiently or stably. Although the transient expression system has the benefits of rapid production, it is more suitable for short-term protein production and the initial phase of antibody testing. Meanwhile, a stable expression system is the go-to for long-term mass protein production because of its ability to express proteins in a reproducible, scalable, and reliable manner with no batch-to-batch variation.
Elucidation Of Erk1/2/C-Myc/P53 Signaling Pathways Involved In Andrographolide-Induced Antiproliferative Activity In Human Glioblastoma Dbtrg-05mg Cell Line
(2023-03)
Nurul Syamimi Binti Othman
The whole study evaluates andrographolide's anticancer effectiveness and potential molecular pathways using a glioblastoma multiforme (GBM) cell line.
Evaluation Of Toxicity, Pharmacokinetic And Clastogenicity Profile Of Ethyl 2-[4[(Piperidin-1-yl) Phenyl]-1h-benzimidazole-5-carboxylate (Bzd9l1), Novel Sirtuin Inhibitor
(2022-05)
Lee, Yeuan Ting
The mammalian sirtuins (SIRTs) have been linked to various diseases including cancer, diabetes, neurodegenerative and cardiovascular diseases. Thus, SIRTs are attractive targets for the development of pharmaceuticals. The lack of potential SIRT inhibitors in cancer clinical trials highlights the need to develop a potent SIRT modulator as an anti-cancer therapeutic. Studies in the lab have highlighted the capability of a lead sirtuin inhibitor (BZD9L1) to reduce colorectal tumour growth in vitro and in vivo by modulating different cancer pathways in colorectal cancer with different mutation profiles. Also, synergistic effects of BZD9L1 in in vitro assays and tumour xenograft study when used in adjunct with 5- Fluorouracil (5-FU) further support the promising therapeutic effects of BZD9L1 as anticancer regime. Nevertheless, the toxicology profile of this compound remains unknown. To our knowledge, no prior work has reported the regulation of SIRTs in the liver and kidney by a small molecule inhibitor in a drug toxicity study. Therefore, this project aims to determine the toxicology, molecular regulation of toxicity, pharmacokinetics and clastogenicity profiles of BZD9L1 in in vitro or in vivo models.
Exploring The Immunomodulation Profiles Of The Thp-1 Human Macrophage-derived Cell Line Mediated By Shigella Flexneri
(2022-06)
Shabani, Nor Raihan Mohammad
Shigellosis is the primary cause of severe diarrhea, especially among children less than five years old. The occurrence of multi-drug resistant strains of Shigella species has contributed to the ineffectiveness of the existing treatment. Vaccination has become an essential requirement for preventing shigellosis. The invention of an epitope-based vaccine has directed the development of a new vaccine design model. Macrophages are specialized APCs that process antigens and then present the processed antigens to T-cells through HLA molecules. The immunomodulatory profiles of the macrophages infected by the clinical strains of S. flexneri 2a have remained undefined. THP-1-derived macrophages, as the model of human macrophages, were infected independently with mild and virulent strains of S. flexneri 2a at standard growth conditions. The gene expression level of inflammatory mediators was determined using qPCR, while NO production was measured using a commercial NO assay kit. The significant up-regulation of TNFα, IL-1β, IL-6, IL-12, iNOS, and NO indicates the pro-inflammatory reaction to S. flexneri 2a infection. The virulent strain also markedly increased the expression levels of the anti-inflammatory cytokine mRNAs. HLA class II gene expression was also investigated to extend the immunopeptidomics study and found the significant up-regulation of HLA class II by the Shigella-infected macrophages. LC-MS/MS-based immunopeptidomics strategy was employed to identify the peptides that can be used as potential candidates for the epitope-based Shigella vaccine development
Genomic Expression Profile Of Human Papillomavirus 16 And 18 – Associated Pre-cancerous Lesions And Cancer Of The Cervix
(2022-06)
Balasubramaniam, Shandra Devi
Cervical cancer is one of the most common cancers in women and is caused by high-risk human papillomavirus (HPV) infection. The integration of HPV into host cervical epithelial cells causes genetic alterations and consequent changes in gene expression affecting downstream molecular pathways, leading to the development of cervical cancer. This study aimed to profile the genomic signatures involved in the pathogenesis of HPV 16 and 18 associated pre-cancerous lesions (cervical intraepithelial neoplasia, CIN) and squamous cell carcinoma (SCC) of the cervix, in comparison to the normal cervix. The differential mRNA expression profiles were determined, to evaluate the expression of up-regulated and down-regulated host genes and to evaluate the perturbed molecular pathways involved in cervical carcinogenesis. In this study, 29 formalin-fixed paraffin-embedded (FFPE) tissues including low-grade CIN (LGCIN), high-grade CIN (HGCIN), SCC, and normal cervix were screened for HPV 16 and HPV 18 using immunohistochemistry and qRT-PCR method. Of these, 9 HPV-positive (3 LGCIN, 3 HGCIN, 3 SCC) and 3 normal cervix (HPV-negative) tissue samples were microdissected to obtain regions of interest in the squamous epithelium before RNA extraction. Transcriptomic profiling was performed using the Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0) and the nCounter® PanCancer Pathway Array, Nanostring to identify differentially expressed genes (DEGs) and significantly associated pathways in each stage of cervical cancer development.
Identification Of Antimalarial Compounds And Their Mode Of Actions
(2022-10)
Mahmud, Fauze
Malaria is one of the most significant infectious diseases in the tropics, claiming around half-million lives annually, mainly due to Plasmodium falciparum (P. falciparum) infections. To date, P. falciparum has developed full or partial resistance towards all three classes of antimalarials, including against the artemisinin. Hence, it may jeopardize the efficacy of the current antimalarial treatment regimen, the Artemisinin Combination Therapy (ACT), in the near future. Therefore, there is an utmost need to identify a new antimalarial compound with a novel mode of action (MoA). This study mainly focused on identifying antimalarial compounds from soil microorganisms isolated from Malaysia and Japan. The crude extracts of these strains showed potent antimalarial activity in a preliminary assay against Pf 3D7 (wild type). The crude extracts of Malaysian strains also inhibited the activity of human GSK-3β (75 % similar to P. Falciparum GSK-3 (Pf GSK-3)).
Identification Of Selected Ageing-Related Proteins And The Characterisation Of Ptc4 In Yeast
(2022-12)
Lee, Jee Whu
Ageing-related proteins play different roles in cellular processes such as regulating stress response, apoptosis, ubiquitin-proteasome system and signal transduction pathways amongst many others. Hundreds of ageing-related proteins have been revealed, but the roles of most of these ageing-related proteins are still unknown. In this study, the standardised chronological life span (CLS) assay, stress assay and growth assay were established. The CLS-extending effects of the ageing-related proteins, Ptc4, Zwf1, Sme1 and Sod1 selected from previous chronological life span (CLS) screen were validated through standardised colony forming unit (CFU) assay and the deletions of the selected ageing-related genes resulted in decreased CLS in yeast. Ptc4 contributes to oxidative stress tolerance in standardised oxidative stress assay and extends CLS the most among the selected ageing-related proteins. Zwf1 and Sod1 that contribute to oxidative stress tolerance in young cells, did not show oxidative stress tolerance in cells after prolonged ageing, suggesting the loss of oxidative stress tolerance capability after prolonged ageing. Besides, Ptc4 and Zwf1 promoted cell proliferation during cell growth upon protein overexpression in standardised growth assays, suggesting their involvement in cell division or growth pathways. Furthermore, loss of Ptc4 could still promote cell proliferation while loss of Zwf1 did not affect rate of cell proliferation. As Ptc4 could promote cell proliferation, the effects of other yeast type 2C protein phosphatases (PP2Cs) on rate of cell proliferation were also investigated in standardised growth assays.
In Vitro And In Vivo Studies On The Complementary Effects Of Standardized Ethanolic Orthosiphon Stamineus Extract On Pancreatic Cancer Cells
(2018-03)
Salem Yehya, Ashwaq Hamid
Pancreatic cancer is globally known as the fourth most lethal cancer in the world. Despite increasing understanding in tumor biology; the efficiency of treatment in pancreatic cancer remains challenging as patients demonstrate resistance to standard treatments. Although the first line agent, gemcitabine has produced clinical response to treat pancreatic cancer, the prognosis remains dismal.
Inhibitory Effects Of Andrographolide In Pc-3 Cell Line And The Induction Of Apoptosis Via The Involvement Of Caspases 3, 8 And 9
(2023-04)
Manimaran, Janany
Andrographolide is a labdane diterpenoid isolated from the plant Andrographis paniculata. This substance has numerous medicinal uses, notably anticancer effects. A previous study has revealed that andrographolide inhibits the growth of lung, brain, colon, and breast cancer cells. Due to a lack of research, the knowledge of andrographolide's anti-cancer effects on prostate cancer cells is relatively poor. In the current study, andrographolide was assessed on PC-3 cells, an aggressive androgen-independent prostate cancer cell line. Cytotoxicity analysis is vital in drug discovery research for assessing the biocompatibility of the drug being used on cancer cells. This work used the WST-1 assay to determine the cell survival of PC-3 cancer cells and Hs27 normal cells exposed to varying doses of andrographolide (0-200 μM). The results indicate that andrographolide dose-dependently suppresses the viability of PC-3 cells but not Hs27 cells. The NCI considers the LC50 value of 26.42 μM (after 48 hours of incubation) is acceptable. For the first time in this study, three different concentrations of andrographolide were used: control, half LC50, and LC50 (0, 13.21, and 26.42 μM) in all subsequent analyses. Metastasis is essential for the disease to progress; hence, the scratch assay and the transwell invasion assay were used to test andrographolide on PC-3 cells.
Isolation, Characterization, And Production Of Recombinant Monoclonal Antibodies Against Rnie For Development Of A Strongyloides Antigen Detection Assay
(2022-05)
Balachandra, Dinesh
Strongyloides stercoralis is a soil-transmitted helminth that causes strongyloidiasis. It is estimated to infect more than 600 million people worldwide. Asymptomatic chronic infections in immunocompromised people can lead to fatal hyperinfection. Serodiagnosis by detecting specific IgG antibodies can be challenging due to potential cross-reactivity with infections by other parasites. An antigen detection assay, a direct detection method, can help the diagnosis and is useful for post-treatment follow-up. This study used phage display technology to produce recombinant monoclonal antibodies (rMAb) against NIE recombinant protein (rNIE) and develop a Strongyloides antigen detection test. rNIE is an established protein for the diagnosis of strongyloidiasis. rNIE was expressed, purified, and then used to select rMAb candidates via biopanning of an immune helminth phage display library. It isolated of 104 ELISA-positive clones and sequence analysis showed that 30 clones had full-length light and heavy chains. Four unique gene families were identified, i.e., IgHV3-LV6 (86.66%), IgHV1-LV3 (3.33%), IgHV5-KV3 (3.33%), and IgHV3-LV3 (6.66%). Randomly, one representative clone from each gene family was selected for further studies, i.e., (a) rMAb5 representing IgHV1-LV3, (b) rMAb6 representing IgHV3-LV6, (c) rMAb14 representing IgHV5-KV3, and (d) rMAb23 representing IgHV3-LV3. The rMAb gene sequences from the phage display vector were subcloned into the pET51b+ expression vector and transformed into Escherichia coli Shuffle T7 Express host cell.
Mechanism Of Action Of A Benzimidazole Sirtuin Inhibitor, Bzd9l1, In Colorectal Cancer
(2022-08)
Tan, Yi Jer
Sirtuins (SIRTs) are NAD+-dependent deacetylases that is implicated in various epigenetic diseases including cardiovascular and neurodegenerative diseases, diabetes, aging and cancer. The emergence of SIRTs as therapeutic targets and limitations of existing SIRT inhibitors led to the discovery of a novel SIRT inhibitor: BZD9L1. As testing of the effects of BZD9L1 on the enzymatic activities of SIRTs have been hampered by the availability of commercial kits, BZD9L1 interactions on SIRTs were studied using molecular modelling and docking studies. Molecular docking studies revealed that BZD9L1 may bind to SIRT1-3, 6 and 7 with similar confirmation but with different affinities, thereby expanding the therapeutic potential of BZD9L1 in metabolic diseases. In addition, in silico approaches were deployed to further elucidate BZD9L1-modulated mechanisms based on existing experimental data. In silico analysis of BZD9L1-regulated targets showed that the proliferation and apoptosis of HCT 116 cells may be due to p53-dependent signalling pathways. BZD9L1 was found to be effective against different cancer cell lines especially colorectal cancer (CRC), where its first-line chemotherapy regimen 5-fluorouracil (5-FU) often result in treatment failure due to drug insensitivity and severe side effects. As current efforts to overcome these boundaries involved sensitizing tumours through adjuvant treatments, this project also aims to provide novel insights into the potential development of BZD9L1 as an adjuvant to 5-FU in CRC therapy using in vitro and in vivo models. The combination of BZD9L1 and 5-FU was found to be more effective against HCT 116 CRC cell line in reducing cell viability and survival compared to sole treatment via synergistic effect.
Mechanism Of Polyalthia Longifolia (Sonn.) Thwaites Pol Yphenols Action In Hela Cells In Relation To Microrna Regulation
(2017-05)
Vijayarathna Soundararajan
Polyalthia longifolia (Sonn.) Thwaites is a lofty, evergreen tree that extensively known for its medicinal beneficial in treating various ailments. Though P. longifolia ethnomedicinal benefits had been recognised, studies concerning its anticancer and apoptotic cell death activity in relation to microRNAs (miRNA) and their mechanism had never been studied in detail.
Modifications Of Nk-92mi Cell Line Through Third Generation Lentiviral Vector
(2023-01)
Chin Ding Sheng
In this study, NK-92MI cell line was transduced with RRL lentivirus carrying the genes for either intracellular GFP or surface CD16 receptor, with the addition of polybrene polycation. Results showed that GFP expression averaged at 3.49 % without polybrene and averaged at 7.18 % when polybrene was added. Extracellular CD16 expression averaged at 1.03 % without polybrene and increased to an average of 3.12 % with polybrene. This study demonstrated the feasibility of using the third generation RRL lentiviral vector for NK cell transduction.
Production And Characterization Of Recombinant Monoclonal Antibodies Against Toxocara Antigens
(2023-07)
Mohd Baharudeen, Zamrina
Toxocariasis is a neglected zoonotic parasitic disease caused by intestinal parasitic roundworms of dogs (Toxocara canis) and cats (Toxocara cati). Human toxocariasis has a global distribution and affects mostly people who are socioeconomically deprived. Clinicians face problems in diagnosing human toxocariasis since the signs and symptoms are non-specific and similar to other helminthic infections. Most serodiagnostic methods for toxocariasis detection rely on IgG antibody-based assays using native antigen of T. canis, however the drawback is the lack of high diagnostic specificity due to cross-reactivity with antibodies to other helminths. Additionally, IgG is a long-lasting antibody, thus IgG assays may also detect past (cured) infections. Thus, there is a need to improve the serodiagnosis of toxocariasis and one good way is by developing an antigen detection assay. To develop such an assay, recombinant monoclonal antibodies (rmAbs) to Toxocara excretory-secretory (TES) antigens can be used as the capture reagent. In this study, two recombinant proteins namely T. canis (rTES-26) and T. cati (rTES-120 cati), were expressed and purified. The isolation of mAbs to the recombinant proteins was performed via biopanning using previously produced helminth phage display immune library. Five mAbs against rTES-26 antigen were successfully isolated. However, mAbs against rTES-120 cati antigen showed incomplete scFv sequences; hence, further analysis could not be performed. Henceforth, only the mAbs of the rTES-26 antigen were characterized based on gene family, length of sequence and amino acid distribution.

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