Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play Important roles in innate immunity In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5'-untranslated region (UTR) of 101 bp and a 3'-UTR of 164 bp The deduced amino acid sequence of SmLec1 shares 34%-38% overall identities with the C-type lectins of several fish species In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus uncle The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 degrees C) Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection (C) 2010 Elsevier Ltd. All rights reserved

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

海洋藻类功能性蛋白质的纯化、鉴定及其应用的研究

使用膨化柱和离子交换或羟基磷灰石柱层析相结合的方法，分别从多管藻、坛紫菜及钝顶螺旋藻中分离纯化了R-藻红蛋白溶液和C-藻蓝蛋白。光谱检测及电泳分析结果证明完全符合经典的藻胆蛋白纯度标准。彭化床最突出的优点是克服了常规分离方法堵塞色谱柱的难题，纯化速度快、产量高、不需要常规色谱方法所要求的填料的平衡及粗提液的预处理，仅需一步操作就可以得到满足一般食品添加剂纯度要求的藻胆蛋白，极大地简化了后续的纯化程序，减少了分离纯化的步骤和时间，而其产率及纯度均高于常规的藻胆蛋白分离方法。这同时也降低了藻胆蛋白分离纯化的成本。 本文通过戊二醛或环氧氯丙烷交联的方法，合成了四种壳聚糖-氨基酸共聚小球。并选取吸附性好的戊二醛交联孔球和戊二醛交联微球系统测定了其对R-藻红蛋白和C-藻蓝蛋白的吸附和缓释性能。 纯化了藓羽藻中与其细胞器团聚密切相关的一种凝集素并进行了部分性质的鉴定。N端前15个氨基酸序列及LC-ESI-MS质谱分析结果证明此凝集素属于一种新的蛋白质族。实验证明，凝血活性与细胞器团聚活性并不完全依赖于此凝集素分子相同的结构域。 通过异双功能试剂SPDP处理藓羽藻凝集素使之衍生化，DTT处理R-PE在其分子内引入外源巯基，然后将活化的R-藻红蛋白与凝集素进行交联反应。交联产物经凝胶过滤纯化并检测，但电泳及荧光显微镜检测结果并不能证明交联探针的成功制备。

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd tauri and Pd strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminat domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species. (C) 2007 Elsevier Ltd. All rights reserved.

cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the Giant Panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E. coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

Novas Insulinas

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.

A variant of SCID with specific immune responses and predominance of gamma delta T cells.

We describe here a patient with a clinical and molecular diagnosis of recombinase activating gene 1-deficient (RAG1-deficient) SCID, who produced specific antibodies despite minimal B cell numbers. Memory B cells were detected and antibodies were produced not only against some vaccines and infections, but also against autoantigens. The patient had severely reduced levels of oligoclonal T cells expressing the alphabeta TCR but surprisingly normal numbers of T cells expressing the gammadelta TCR. Analysis at a clonal level and TCR complementarity-determining region-3 spectratyping for gammadelta T cells revealed a diversified oligoclonal repertoire with predominance of cells expressing a gamma4-delta3 TCR. Several gammadelta T cell clones displayed reactivity against CMV-infected cells. These observations are compatible with 2 non-mutually exclusive explanations for the gammadelta T cell predominance: a developmental advantage and infection-triggered, antigen-driven peripheral expansion. The patient carried the homozygous hypomorphic R561H RAG1 mutation leading to reduced V(D)J recombination but lacked all clinical features characteristic of Omenn syndrome. This report describes a new phenotype of RAG deficiency and shows that the ability to form specific antibodies does not exclude the diagnosis of SCID.

Barnacle cement: a polymerization model based on evolutionary concepts.

Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues.

Similar T-cell immune responses induced by group M consensus env immunogens with wild-type or minimum consensus variable regions.

Consensus HIV-1 genes can decrease the genetic distances between candidate immunogens and field virus strains. To ensure the functionality and optimal presentation of immunologic epitopes, we generated two group-M consensus env genes that contain variable regions either from a wild-type B/C recombinant virus isolate (CON6) or minimal consensus elements (CON-S) in the V1, V2, V4, and V5 regions. C57BL/6 and BALB/c mice were primed twice with CON6, CON-S, and subtype control (92UG37_A and HXB2/Bal_B) DNA and boosted with recombinant vaccinia virus (rVV). Mean antibody titers against 92UG37_A, 89.6_B, 96ZM651_C, CON6, and CON-S Env protein were determined. Both CON6 and CON-S induced higher mean antibody titers against several of the proteins, as compared with the subtype controls. However, no significant differences were found in mean antibody titers in animals immunized with CON6 or CON-S. Cellular immune responses were measured by using five complete Env overlapping peptide sets: subtype A (92UG37_A), subtype B (MN_B, 89.6_B and SF162_B), and subtype C (Chn19_C). The intensity of the induced cellular responses was measured by using pooled Env peptides; T-cell epitopes were identified by using matrix peptide pools and individual peptides. No significant differences in T-cell immune-response intensities were noted between CON6 and CON-S immunized BALB/c and C57BL/6 mice. In BALB/c mice, 10 and eight nonoverlapping T-cell epitopes were identified in CON6 and CON-S, whereas eight epitopes were identified in 92UG37_A and HXB2/BAL_B. In C57BL/6 mice, nine and six nonoverlapping T-cell epitopes were identified after immunization with CON6 and CON-S, respectively, whereas only four and three were identified in 92UG37_A and HXB2/BAL_B, respectively. When combined together from both mouse strains, 18 epitopes were identified. The group M artificial consensus env genes, CON6 and CON-S, were equally immunogenic in breadth and intensity for inducing humoral and cellular immune responses.