Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.

Plasmodium knowlesi circumsporozoite protein: genetic characterisation and predicted antigenicity of the central repeat region

@#Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif “AGQPQAQGDGANAGQPQAQGDGAN” has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

Bioinformatics characterization of Plasmodium knowlesi apical membrane antigen 1 (PkAMA1) for multi-epitope vaccine design

@#Malaria caused by Plasmodium knowlesi species has become a public health concern, especially in Malaysia. Plasmodium knowlesi parasite which originates from the macaque species, infects human through the bite of the Anopheles mosquitoes. Research on malaria vaccine has been a continuous effort to eradicate the malaria infection, yet there is no vaccine against P. knowlesi malaria to date. Apical membrane antigen 1 (AMA1) is a unique surface protein of all apicomplexan parasites that plays a crucial role in parasite-host cell invasion and thus has been a long-standing malaria vaccine candidate. The selection of protective epitopes in silico has led to significant advances in the design of the vaccine. The present study aimed to employ bioinformatics tools to predict the potential immunogenic B- and T-cell epitopes in designing malaria vaccine targeting P. knowlesi AMA1 (PkAMA1). B-cell epitopes were predicted using four bioinformatics tools, i.e., BepiPred, ABCpred, BcePred, and IEDB servers whereas T-cell epitopes were predicted using two bioinformatics servers, i.e., NetMHCpan4.1 and NetMHCIIpan-4.0 targeting human major histocompatibility complex (MHC) class I and class II molecules, respectively. The antigenicity of the selected epitopes computed by both B- and T-cell predictors were further analyzed using the VaxiJen server. The results demonstrated that PkAMA1 protein encompasses multi antigenic regions that have the potential for the development of multi-epitope vaccine. Two B- and T-cell epitopes consensus regions, i.e., NSGIRIDLGEDAEVGNSKYRIPAGKCP (codons 28-54) and KTHAASFVIAEDQNTSY RHPAVYDEKNKT (codons 122-150) at domain I (DI) of PkAMA1 were reported. Advancement of bioinformatics in characterization of the target protein may facilitate vaccine development especially in vaccine design which is costly and cumbersome process. Thus, comprehensive B-cell and T-cell epitope prediction of PkAMA1 offers a promising pipeline for the development and design of multi-epitope vaccine against P. knowlesi.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHCⅡ and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Prediction of B cell Epitopes and HLA I Restricted CTL Epitopes Derived from Tumor Antigen SCCAg / 现代医院

Ojbective To predicte the HLAⅠrestricted CTL epitopes and B cell antigen epitopes derived from tumor antigen SCCAg. Methods The linear B cell epitopes and conformational B cell epitopes of tumor antigen SCCAg were predicted by Ellipro program. In addition, the HLAⅠrestricted CTL epitopes of SCCAg were predicted by NetCTL, Prot-Param and so on. Results B cell epitopes analysis revealed that SCCAg had 10 potential linear B cell epitopes and 5 conformational B cell epitopes; Combined with peptide HLAⅠbinding, proteasomal C - terminal cleavage and TAP transport efficiency, the NetCTL predicts that multiple HLAⅠrestricted CTL epitopes were present in the tumor antigen SCCAg. Conclusion The B cell epitopes and HLAⅠrestricted CTL epitopes can be predicted by multiple methods, which may lay the foundation for the further research on immunotherapy for targeting SCCAg.

Bioinformatics characterization of envelope glycoprotein from Kyasanur Forest disease virus

Background & objectives: Kyasanur Forest disease (KFD) is a febrile illness characterized by haemorrhages and caused by KFD virus (KFDV), which belongs to the Flaviviridae family. It is reported to be an endemic disease in Shimoga district of Karnataka State, India, especially in forested and adjoining areas. Several outbreaks have been reported in newer areas, which raised queries regarding the changing nature of structural proteins if any. The objective of the study was to investigate amino acid composition and antigenic variability if any, among the envelope glycoprotein (E-proteins) from old and new strains of KFDV. Methods: Bioinformatic tools and techniques were used to predict B-cell epitopes and three-dimensional structures and to compare envelope glycoprotein (E-proteins) between the old strains of KFDV and those from emerging outbreaks till 2015. Results: The strain from recent outbreak in Thirthahalli, Karnataka State (2014), was similar to the older strain of KFDV (99.2%). Although mutations existed in strains from 2015 in Kerala KFD sequences, these did not alter the epitopes. Interpretation & conclusions: The study revealed that though mutations existed, there were no drastic changes in the structure or antigenicity of the E-proteins from recent outbreaks. Hence, no correlation could be established between the mutations and detection in new geographical areas. It seems that KFDV must be present earlier also in many States and due to availability of testing system and alertness coming into notice now.

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein: An in silico approach

Objective To predict immunogenic promiscuous T cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools. To date, no epitope data are available for the Zika virus in the IEDB database. Methods We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks. A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using PorPred1 and ProPred immunoinformatic algorithms respectively. The antigenicity predicted score was also calculated for each predicted epitope using the VaxiJen 2.0 tool. Results By using ProPred1, 23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus. The greatest number of MHC class I binding epitopes were projected within the NS5 (21%), followed by Envelope (17%). For MHC class II, greatest number of predicted epitopes were in NS5 (19%) followed by the Envelope, NS1 and NS2 (17% each). A variety of epitopes with good binding affinity, promiscuity and antigenicity were predicted for both the HLA classes. Conclusion The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates, which will be able to induce a broad range of immune responses in a heterogeneous HLA population. However, our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

Prediction of promiscuous T-cell epitopes in the Zika virus polyprotein: An in silico approach

OBJECTIVE@#To predict immunogenic promiscuous T cell epitopes from the polyprotein of the Zika virus using a range of bioinformatics tools. To date, no epitope data are available for the Zika virus in the IEDB database.@*METHODS@#We retrieved nearly 54 full length polyprotein sequences of the Zika virus from the NCBI database belonging to different outbreaks. A consensus sequence was then used to predict the promiscuous T cell epitopes that bind MHC 1 and MHC II alleles using PorPred1 and ProPred immunoinformatic algorithms respectively. The antigenicity predicted score was also calculated for each predicted epitope using the VaxiJen 2.0 tool.@*RESULTS@#By using ProPred1, 23 antigenic epitopes for HLA class I and 48 antigenic epitopes for HLA class II were predicted from the consensus polyprotein sequence of Zika virus. The greatest number of MHC class I binding epitopes were projected within the NS5 (21%), followed by Envelope (17%). For MHC class II, greatest number of predicted epitopes were in NS5 (19%) followed by the Envelope, NS1 and NS2 (17% each). A variety of epitopes with good binding affinity, promiscuity and antigenicity were predicted for both the HLA classes.@*CONCLUSION@#The predicted conserved promiscuous T-cell epitopes examined in this study were reported for the first time and will contribute to the imminent design of Zika virus vaccine candidates, which will be able to induce a broad range of immune responses in a heterogeneous HLA population. However, our results can be verified and employed in future efficacious vaccine formulations only after successful experimental studies.

Prediction of the secondary structure and B-cell epitopes of Mycoplasma suis ORF5 protein / 中国人兽共患病学报

The objective of the study is to predict the spatial structure and B‐cell epitopes of Mycoplasma suis ORF5 pro‐tein .The secondary structure ,hydrophilicity ,flexible region ,antigenic index and surface probability were analyzed and predic‐ted by the Protean module in DNAStar software and B Cell Epitope Prediction Tools of IDEB ,then B‐cell epitopes were predic‐ted by aggregate analysis .Results showed that the secondary structure of Mycoplasma suis ORF5 protein was relatively regu‐lar ,in which the potential B cell antigenic epitopes were located at GGVDGGRD ,GMRLPEDSR ,and EGHPDLESAR .The methods of prediction of the secondary structure and B‐cell epitopes of Mycoplasma suis ORF5 protein may provide a new method for the study of M .suis immunogenicity ,and provides a new idea for the study on immunogenicity of pathogenic micro‐organisms .

B cell line epitopes prediction of human cytomegalovirus glycoprotein B / 军事医学

Objective To predict the B cell line epitopes of human cytomegalovirus glycoprotein (gB)by analyzing its structure and physicochemical properties using bioinformatics approaches .Methods Based on the sequence of the HCMV gB,the probable B cell epitopes are predicted using two online prediction programs and DNAstar software .Meanwhile,the tertiary structure of gB was constructed by homologous modeling with the assistance of SWISS -MODEL server to rule out im-possible B cell epitopes .Results and Conclusion The B cell line epitopes of gB are predicted , which provides a theoreti-cal basis for further verification of gB immunodominant epitopes and screening the source plasma with high HCMV IgG titer .

Prediction of the secondary structure and B cell epitopes for the Izumo protein of Homo Sapiens / 中国免疫学杂志

Objective:To predict and analyze the secondary structure and B cell epitopes of Izumo protein.Methods: The secondary structure and flexible regions of Izumo protein were predicted by the methods of Chou-Fasman,Gamier-Robson and Karplus-Schulz.Moreover,hydrophilicity plot,surface probability plot and antigenic index of Izumo protein were predicted by the methods of Kyte-Doolitde,Emini and Jameson-Wolf,respectively.Results: Izumo protein contained moreαhelix regions.There were several centers ofαhelix in the regions of 6-17,30-40,88-99,103-120,153-160,173-188,249-260,283-297,334-338 and 339-346 of Izumo protein,and several centers of βsheet in the regions of 21-25,198-200,245-248 and 320-323.Moreover,many distinct B cell epitopes in Izumo protein possibly localized in the regions of 3642,62-66,94-99,118-122,129-132,151-154,161-164,173-177,205-208,212-216,256-265,271-276,283-288,314-318 and 336-350.Conclusion:These results are helpful for identification of the dominant B cell epitopes and the functional domains of Izumo protein.

Prediction of the B cell epitopes of human heparanase protein and determination of their immunogenicity / 中华微生物学和免疫学杂志

Objective To predict the secondary structure and the B cell epitopes of human heparanase protein, and to identify its immunogenicity. Methods The flexible regions of secondary structure and the B cell epitopes of human heparanase amino acid sequence were predicted by DNAStar and Bcepred software. The multiple antigenic peptides (MAP) of the epitopes were synthesized in 8-branch form. Rabbits were immunized with the 8-branch MAPs mixed with a universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163-171 ). The immunogenicity of the synthesized peptides was evaluated by ELISA, Western blot and immunohistochemistry. Results Amino acid 1 -15 ( MAP1), 279-293 (MAP2) and 175-189(MAP3) of large-subunit of human heparanase protein was predicted as the most potential epitopes of human heparanase protein. All the three synthesized MAPs induced high titer of antibodies. ELISA, Western blot and immunohistochemistry analysis showed all the three MAPs could produce high titer serum antibodies, antibodies induced by MAP1 and MAP2 had high specific binding activity , and MAP2 antibody showed the strongest binding activity with liver cancer tissues. Conclusion The large-subunit No. 1-15, 279-293 amino acid of human heparanase protein may be the B cell preponderant epitopes and the strongest immunogenicity may be No. 279-293 peptide, which provided a theoretic basis for the antibody and vaccine development of heparanase subunit peptide.

Prediction of the Secondary Structure and B Cell Epitopes of DMO and DMT Protein in Oreochromis aureus / 中国生物工程杂志

The secondary structure of the protein of DM0 and DMT in Oreochromis aureus were predicted by the methods of Garnier-Robson, Chou-Fasman and Karplus-Schulz based on the amimo acid sequences of DM0 and DMT. And Hydrophilicity plot, Surface probability and Antigenic index for DM0 and DMT protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. Combined the results according to these methods, the B cell epitopes for DM0 and DMT protein were predicted. The results demonstrated that there were some centers of?-helix in the DM0 protein' s N- terminal No. 80 - 112, 144 -147, 193- 194, 251 - 255, 260 - 269 and No. 279- 283, and in the DMT protein' s N-terminal No. 61 -86, 98 - 105, 140 - 146, 239 -241 and No. 269 -273. And there are some centers of?-sheet in the DM0 protein' s N-terminal No. 59 -61, 69 -70, 148 - 150 and No. 383 -390, and in the DMT protein's N-terminal No. 125 -129, 207-213, 255-264 and No. 281-284. Furthermore, the DMO protein' s N-terminal No. 40-41,44 -45, 50-51, 128-129, 189-192, 204-207, 216-222, 226-233, 244-246, 298 - 299 and No. 323 -326, and the DMT protein' s N-termianl No. 12 - 13, 26 - 27, 43 - 44, 58 - 60, 93 - 95, 115 - 120, 136 -139 and No. 149 -151 may be the flexible regions. Moreover the B-cell epitopes possibly localized in or nearby the DMO protein's 1 -5, 41 -51, 65-67, 86-89, 98-110, 154-170, 183-203, 205 -248, 258-264, 284 - 291, 293 - 298, 270 - 375, 389 - 392 and No. 402 - 410, and DMT protein' s N-termianl No. 1 - 9, 17 - 28, 77 - 84, 114 - 123 , 131 - 139, 157 - 184 and No. 96 - 207. Theses results are helpful for studies on sex control mechanism of DMO and DMT in Oreochromis aureus.

Prediction of Secondary Structure and B Cell Epitope for Capsid Protein of SVDV / 中国生物工程杂志

The secondary structure of Capsid protein was predicted by the methods of Chou-Fasman,Garnier-Robson and Karplus-Schultz based on the sepuence of capsid protein gene of Swine Vesicular Disease Virus (SVDV) and hydrophilicity. Surface probility plot and antigenic index for capsid protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-wolf, respectively, Combining the results according to these methods, the B cell epitopes for capsid protein of SVDV were predicted. The results showed that there are much flexible region such as coil region and turn region in capsid protein of SVDV, there are more predominant B cell epitopes in VP1 than in VP2 and VP3. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of SVDV.

Evaluation of Differential Antigenic Properties of Selected B - cell Epitopes from the HIV - 1 p24 Protein using Synthetic Peptides / 대한면역학회지

The gag encoded p24 protein of human immunodeficiency virus-1 (HIV-1) is a major constitutent of the viral core, and is also known as one of the most immunodominant antigens in the host immune response against the HIV-1. Based on the neutralizing ability of anti-p24 antibodies as well as their rapid appearance in human serum after viral infection, the development of vaccines and diagnostic tools targeting the p24 protein and anti-p24 antibodies is of great interest. For the characterization of the immunological properties of the HIV-1 p24 protein, in a previous study, putative B-cell epitopes were identified by screening the reactivity of a goat anti-p24 antiserum to a large array of overlapping synthetic peptides covering the whole p24 sequence. Four peptides were identified for their abilities to elicit a strong B-cell response, which sequences comprises the regions p24 (164-182), (202-221), (217-236) and (232-256), respectively. In the present study, the immunogenicity and differential properties of each of these individual epitopes were further characterized. To evaluate the time course of the antibody response, BALB/c mice were immunized with the HIV-1 p24 protein and their serum titers against each of these peptides were determined. The earliest immune response was observed against the p24 (202-221) peptide, which also showed the highest antibody titer against the immunized antigen. Furthermore,. enzyme-linked immunosorbent assay with HIV-1 p24 protein coated microtiter plates revealed that anti-p24 (202-221) antiserum has the most pronounced reactivity against the native p24 protein. Since the p24 (202-221) epitope has also been reported to include a cytotoxic T-lymphocyte epitope, it is suggested that this region might represent a powerful antigenic site responsible for eliciting both T- and B-cell immune response. The possible application of this specific epitope in vaccine development or AIDS diagnosis is discussed.

Design and Construction of G2 Glycoprotein Multi-epitope Antigen Gene of Hantavirus / 环境与健康杂志

Objective To construct the multi-epitope antigen(mea) gene of G2 glycoprotein of Hantavirus SEO type L99 strain.Methods The B cell epitopes was chosen and connected by three-peptide GPG after the amino acid sequence of G2 protein was analyzed and predicted by bioinformatical soft wares.The corresponding gene mea was constructed by overlap PCR,and cloned into the prokaryotic expression plasmid pET32a(+).Results The mea gene was successfully constructed by the five epitopes being chosen.The recombinant plasmid pET32a-mea was acquired by directional cloning.Conclusion The mea and its expression system E.coli BL21/pET32a-mea were constructed for the first time.The foundation was laid for the expression of the mea and its immunological applications.