Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Produção da proteína recombinante Sm15 com potencial para o diagnóstico de pacientes infectados com Schistosoma mansoni

O exame parasitológico por Kato-Katz ainda é considerado o padrão ouro no diagnóstico da esquistossomose mansônica, entretanto, este apresenta baixa sensibilidade para utilização em inquéritos epidemiológicos. Além disso, as técnicas de diagnóstico imunológico, apresentam reações cruzadas com outros helmintos, protozoários e até bactérias como ocorre com a utilização dos extratos brutos do parasita. Nesse sentido, salientamos que Abath et al. identificaram um peptídeo de 15kDa denominado Sm15, que apresentou uma boa reatividade com soros de animais infectados pelo verme e, portanto, possui potencial para abordagens imunoprofiláticas e para testes diagnósticos. Neste estudo obtivemos o polipeptídio recombinante Sm15 em Escherichia coli e verificamos seu potencial para realização do diagnóstico a partir de amostras de soros de pacientes com diferentes manifestações clínicas da esquistossomose. Através de ELISA constatamos que o Sm15 apresentou maior reatividade frente a soros de pacientes esquistossomóticos, quando comparado ao extrato bruto SEA (P=0.0043). O Sm15 ainda demonstrou melhor desempenho ao apresentar maiores valores de sensibilidade, especificidade e área abaixo da curva ROC (P=0.0030). Além disso, o Sm15 foi capaz de diferenciar pacientes esquistossomóticos quanto à forma clínica, aguda ou crônica (P=0.0007). Os resultados obtidos neste estudo indicam, além de ratificar o potencial diagnóstico apresentado pelo polipeptídeo Sm15, que o mesmo poderá ser capaz de gerar uma alternativa de imunodiagnóstico de elevada acurácia, suprindo assim as lacunas existentes com relação aos testes parasitológicos e sorológicos atualmente disponíveis. Além disso, possibilitará o diagnostico precoce da esquistossomose, realização de inquéritos epidemiológicos em áreas de baixa endemicidade, impedindo assim a evolução da doença para formas clínicas de maior gravidade.

The parasitological examination by Kato-Katz still considered the gold standard in the diagnosis of schistosomiasis, however, it has low sensitivity for use in epidemiological surveys. Moreover, the techniques of immunological diagnosis, have cross-reactivity with other helminth, protozoa and even bacteria as occur with the use of crude parasite extracts. In this regard, we note that Abath and colleagues identified a 15kDa peptide termed SM15, which showed good reactivity with sera from animals infected by the worm, and therefore has potential immunoprophylactic and diagnostic testing approaches. In this study we obtained the recombinant polypeptide in Escherichia coli SM15 and check its potential for making the diagnosis from samples of patient sera with different clinical manifestations of schistosomiasis. By ELISA we found that the SM15 showed higher reactivity towards sera from schistosomiasis patients, when compared to the crude extract SEA (P = 0.0043). The SM15 also demonstrated better performance by presenting higher sensitivity, specificity, and area under the ROC curve (P = 0.0030). In addition, the SM15 was able to differentiate schistosomiasis patients about the clinical presentation, acute or chronic (P = 0.0007). The results of this study indicate not only ratifies the diagnostic potential presented by the SM15 polypeptide, that it may be able to generate an immunodiagnostic alternative high accuracy, thereby supplying the gaps in the parasitological and serological tests currently available. Also, it enables the early diagnosis of schistosomiasis, carrying out epidemiological surveys in low endemicity areas, thereby preventing disease progression to more severe clinical forms.

Inhibitory Effect of Recombinant Fibronectin Polypeptide CH50 on Invasion and Metastasis of Melanoma B16 Cells / 华中科技大学学报（医学）（英德文版）

In order to investigate the inhibitory effect and mechanism of recombinant polypeptide CH50 on invasion and metastasis of melanoma B16 cells, the recombinant polypeptide CH50 was separated and purified by ion exchange chromatographic technique. The melanoma B 16 cells treated with purified CH50 were cultured in vitro, the number was counted at 4, 24, 48 and 72 h and their morphological changes were observed in order to detect their adhesion and spreading abilities. In in vivo study, the melanoma B16 cells were labeled with CFSE and treated with CH50 and then they were injected into mice via mouse-tail veins. After 5 h, the lung tissues were fixed by frozen section. Accumulation and invasion abilities of B 16 cells on lung tissues were observed under the fluorescent microscopy. The results showed that the morphological character of B 16 cells treated with CH50 changed greatly and the number of B16 cells treated with CH50 decreased significantly (P＜0.05). The adhesion and spreading abilities of B16 cells treated with CH50 were weakened obviously and the metastasis foci on lung tissues reduced. It was concluded that the recombinant polypeptide CH50 inhibited invasion and metastasis of melanoma B16 cells on tissues and could be a prospective bio-product in tumor general therapy.

Effect of Polypeptide CH50 on Macrophage Activation in vivo and Its Antitu- mor Function / 中国肿瘤生物治疗杂志

Objective: To study the main features of CH50, a recombinant polypeptide of human fibronectin, to activate macrophages in vivo and its anti-tumor function. Methods: CH50 was injected and IFN-? gene was transfected in mice several kinds of factors produced by marcrophages were determined. The growth of tumor in force was also measured. Results: CH50 could enhance the production of such factors as NO, TNF and IL-1 by macrophages, but the activation of macrophages was rela- tively slow when CH50 was used in vivo alone. CH50 and IFN-? could synergistically activate macrophages rapidly in vivo no matter whether the injection of CH50 or the transfection of IFN-? gene was Performed first.Injection of CH50 alone inhibited the formation of tumor nodes in a dose-dependent manner. There was strong inhibition of low dosage of CH50 on the formation of tu- mor nodes smaller than 1 mm, and high dosage of CH50 on those bigger than 1 mm. A stronger inhibition on the growth of tumor in vivo was obtained by the synergistic effect of CH50 and IFN-?. Conclusion: CH50 and IFN-?, as double-signal factors for activation of macrophages, will be potentially useful in tumor therapy.