scholarly journals Mapping of Escherichia coli H27-Specific Epitope from H-Specific Polypeptides

2001 ◽  
Vol 8 (6) ◽  
pp. 1126-1130
Author(s):  
J. N. Seah ◽  
J. Kwang
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Variable Region ◽  
Antigenic Determinants ◽  
Amino Acid Residues ◽  
Flagellin Gene ◽  
Specific Epitope ◽  
Central Variable Region ◽  
Mab Production ◽  
Antigenic Epitopes

ABSTRACT A murine monoclonal antibody (MAb) reactive to H27 flagellin antigen was produced and characterized. Forty-nine partially purified native H-type flagellins were used to evaluate the specificity of the MAb. The fliC gene of H27 is 1,464 bp in length (487 amino acids [aa]; 50.88 kDa). The central variable region (CVR) of the H27 flagellin gene was defined by comparison with flagellin sequences derived from H8, H34, and H49. To study the distribution of antigenic epitopes, the CVR covering amino acid residues 70 to 457 (388 aa) was dissected into seven overlapping fragments. Fragments carrying the H-type-specific antigenic determinants were identified by H27-specific antiserum. Polyclonal antibodies raised against different H-type flagellin proteins were used to determine the cross-reactive determinants. Three fragments, spanning amino acid residues 240 to 380, which carried the potential H-specific determinants were used for MAb production. A MAb specific to H27 was produced, and the specific epitope was mapped to amino acid residues 330 to 340. In this study, we produced MAbs from predetermined H27-specific polypeptides and used whole flagellin in enzyme-linked immunosorbent assays to circumvent the interference of anti-glutathione S-transferase antibodies. These factors when combined could help to improve the identification of the desired MAb.

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals Identification of antigenic epitopes for Guertu virus nucleocapsid protein

2021 ◽  
Author(s):  
Siyuan Wang ◽  
Xihong Yue ◽  
Alai Shalitanati ◽  
Abulimiti Moming ◽  
Shu Shen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epitope Mapping ◽  
Virus Detection ◽  
Polyclonal Antiserum ◽  
Detection Methods ◽  
Amino Acid Residues ◽  
High Conservation ◽  
Potential Pathogenicity ◽  
Severe Fever ◽  
Antigenic Epitopes

Abstract Guertu virus (GTV), a novel tick-borne virus with potential pathogenicity, was first isolated from Dermacentor nuttalli in Xinjiang, China, in 2014. GTV has been shown to infect animal and human cell lines and to be pathogenic in mice. The viral nucleoprotein (NP) is the most conserved immunogenic protein. Elucidating the B-cell epitopes (BCEs) in the immunodominant region of the NP is important for the development of virus detection methods and vaccines. In order to identify the minimal motifs of linear BCEs in the NP of the GTV DXM strain, we used an improved biosynthetic peptide (BSP) method to truncate GTV NP into 30 16mer-peptides with 8 overlapping amino acid residues spanning the full length of the protein. The peptides were analyzed by western blot using rabbit anti-GTV NP polyclonal antiserum, and four positive 16mer-peptides were obtained. The 16mer-peptides were then truncated into 31 8mer-peptides with 7 overlapping amino acid residues and 10mer-peptides with 9 overlapping amino acid residues to screen for BCEs that can react with the rabbit anti-GTV NP polyclonal antiserum. The results showed that there were 6 minimal BCE motifs, namely, Enp1, 88EKYGLVER95; Enp2, 88EKYGLVER95; Enp3, 162TTKILMEA169; Enp4, 187GASKAEVY194; Enp5, 191AEVYNSFR198; and Enp6, 236ETAAAAYRNL245. Positive sheep sera could recognize all six BCEs with anti-GTV antibodies. The BCEs were aligned with the sequences of eight representative severe fever with thrombocytopenia syndrome phlebovirus strains from different countries and regions that were evolutionarily closely related to GTV. The sequence identity of the BCEs ranged from 80–100%, thus showing high conservation. The fine epitope mapping of GTV NP can be used to explore the biological and immunological properties of GTV NP antigens and serve as basic data for the development of multi-epitope detection reagents and vaccine design for GTV.

scholarly journals Possible role for a human adenovirus in the pathogenesis of celiac disease.

1984 ◽  
Vol 160 (5) ◽  
pp. 1544-1557 ◽  
Author(s):  
M F Kagnoff ◽  
R K Austin ◽  
J J Hubert ◽  
J E Bernardin ◽  
D D Kasarda
Keyword(s):  
Celiac Disease ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Monozygotic Twins ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Data Bank ◽  
Antigenic Determinants ◽  
Amino Acid Residues ◽  
Primary Amino

Celiac disease in humans is activated by the dietary ingestion of wheat, rye, triticale, barley, and possibly oats. Gliadins in wheat and similar proteins in the other grains are known to activate disease in susceptible individuals. There is a striking association between celiac disease and HLA-B8, -DR3 and/or -DR7, and -DC3. Nonetheless, less than 0.2% of individuals with those serologic HLA specificities develop celiac disease and disease is not always concordant among monozygotic twins. We propose that additional environmental factors may be important in the pathogenesis of celiac disease. To investigate that possibility, we examined a data bank of protein sequences for other proteins that might share amino acid sequence homologies with A-gliadin, an alpha-gliadin component known to activate celiac disease and whose complete primary amino acid sequence is known. These studies demonstrate that A-gliadin shares a region of amino acid sequence homology with the 54-kD E1b protein of human adenovirus type 12 (Ad12), an adenovirus usually isolated from the intestinal tract. The region spans 12 amino acid residues, includes 8 residue identities and an identical pentapeptide, and is hydrophilic in both proteins. Antibody reactive with the 54-kD Ad12 E1b protein cross-reacts with A-gliadin, a 119 amino acid cyanogen bromide peptide of A-gliadin that spans the region of homology and a synthetic heptapeptide of A-gliadin from within the region of homology. We suggest that an encounter of the immune system with antigenic determinants produced during intestinal viral infection may be important in the pathogenesis of celiac disease.

scholarly journals The complete amino acid sequence of a prototype immunoglobulin-λ light-chain-type amyloid-fibril protein AR

1981 ◽  
Vol 195 (3) ◽  
pp. 561-572 ◽  
Author(s):  
K Sletten ◽  
J B Natvig ◽  
G Husby ◽  
J Juul
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Amyloid Fibril ◽  
Variable Region ◽  
Amino Acid Residues ◽  
Variable Regions ◽  
Light Chain Type ◽  
Amyloid Fibril Protein ◽  
Chain Type

The amino acid sequence of an amyloid-fibril protein of immunoglobulin light-chain type (AL) was elucidated. The sequence determination involved digesting the protein with trypsin, thermolysin and pepsin. The protein was found to consist of 154 amino acid residues and is thus missing about half of the constant region of a light chain. A certain heterogeneity in the length of the polypeptide was observed in the C-terminal region. The amino acid sequence from CDR (complementary-determining region) 1 and FR (framework region) 3 indicated an oligoclonal origin of the protein. By comparing the primary structure of protein AR with other lambda- and even kappa-chains, it was revealed that protein AR had an insertion of two residues of aspartic acid, namely residues 68 and 69, which has not been reported previously in light chains. The overall sequence homology in the variable region showed that protein AR is more similar to V lambda V than to the other subgroups [Kabat, Wu & Bilofsky (1979) Variable regions of Immunoglobulin Chains, Medical Computer Systems, Bolt, Beranek and Newman, Cambridge, MA].

scholarly journals Amino acid-sequence variability at the N-terminal extra piece of mouse immunoglobulin light-chain precursors of the same and different subgroups

1976 ◽  
Vol 157 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Y Burstein ◽  
I Schechter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Light Chain ◽  
Variable Region ◽  
Amino Acid Residues ◽  
Free System ◽  
Methionine Residue ◽  
N Terminus ◽  
Mouse Myeloma

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-63 mouse myeloma L (light) chain were labelled with six radioactive amino acids: [35S]methionine, [4,5-3H]leucine, [3,4-3H]proline, [3-3H]serine, [4,5-3H]isoleucine or [2,3-3H]alanine. Amino acid-sequence analyses showed that over 90% of the total cell-free product was one homogeneous protein, which corresponds to the MOPC-63 L-chain precursor. In this precursor an extra piece, 20 amino acid residues in length, precedes the N-terminus of the mature L chain. The extra piece contains one methionine residue at the N-terminus, six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13, one proline residue at position 16, and one serine residue at position 18. The closely gathered leucine residues, as well as their abundance (30%), suggest that the extra-piece moiety is hydrophobic. In the precursors, the extra piece is coupled to the variable region of the L chain. Partial sequences of precursors of L chains of the same and different subgroups that were labelled with the above six radioactive amino acids indicate that the extra piece is part of the variable region. Thus the precursors of MOPC-63 and MOPC-321 L chains, which are of the same subgroup, have extra pieces of identical size (20 residues), and so far their partial sequences are also identical (see above). On the other hand, in the precursor of MOPC-41 L chain, which is of a different subgroup, the extra piece is 22 residues in length. Further, the sequence of the MOPC-41 extra piece differs in at least ten positions from sequences of the extra pieces of the precursors of MOPC-63 and MOPC-321 L chains.

scholarly journals Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.

1991 ◽  
Vol 173 (3) ◽  
pp. 665-672 ◽  
Author(s):  
S Fish ◽  
M Fleming ◽  
J Sharon ◽  
T Manser
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Somatic Mutation ◽  
Amino Acid Substitution ◽  
Variable Region ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
V Region ◽  
Mutant Forms ◽  
Selection Of

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.

scholarly journals Molecular Analysis of the Multiple GroEL Proteins of Chlamydiae

2003 ◽  
Vol 185 (6) ◽  
pp. 1958-1966 ◽  
Author(s):  
Karuna P. Karunakaran ◽  
Yasuyuki Noguchi ◽  
Timothy D. Read ◽  
Artem Cherkasov ◽  
Jeffrey Kwee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Developmental Cycle ◽  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
E Coli ◽  
Chaperonin Groel ◽  
Mutant Complementation

ABSTRACT Genome sequencing revealed that all six chlamydiae genomes contain three groEL-like genes (groEL1, groEL2, and groEL3). Phylogenetic analysis of groEL1, groEL2, and groEL3 indicates that these genes are likely to have been present in chlamydiae since the beginning of the lineage. Comparison of deduced amino acid sequences of the three groEL genes with those of other organisms showed high homology only for groEL1, although comparison of critical amino acid residues that are required for polypeptide binding of the Escherichia coli chaperonin GroEL revealed substantial conservation in all three chlamydial GroELs. This was further supported by three-dimensional structural predictions. All three genes are expressed constitutively throughout the developmental cycle of Chlamydia trachomatis, although groEL1 is expressed at much higher levels than are groEL2 and groEL3. Transcription of groEL1, but not groEL2 and groEL3, was elevated when HeLa cells infected with C. trachomatis were subjected to heat shock. Western blot analysis with polyclonal antibodies raised against recombinant GroEL1, GroEL2, and GroEL3 demonstrated the presence of the three proteins in C. trachomatis elementary bodies, with GroEL1 being present in the largest amount. Only C. trachomatis groEL1 and groES together complemented a temperature-sensitive E. coli groEL mutant. Complementation did not occur with groEL2 or groEL3 alone or together with groES. The role for each of the three GroELs in the chlamydial developmental cycle and in disease pathogenesis requires further study.

scholarly journals The primary structure of the variable region of an immunoglobin IV light-chain amyloid-fibril protein (AL GIL)

1988 ◽  
Vol 256 (3) ◽  
pp. 973-980 ◽  
Author(s):  
E M Fykse ◽  
K Sletten ◽  
G Husby ◽  
G G Cornwell
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Primary Structure ◽  
Amyloid Fibril ◽  
Variable Region ◽  
Acceptor Site ◽  
Amino Acid Residues ◽  
Bence Jones Protein ◽  
Sequence Homologies ◽  
Amyloid Fibril Protein

The primary structure of the variable region of an amyloid-fibril protein GIL of immunoglobulin lambda-light-chain origin (AL) was determined. The AL protein obtained from the fibrils in the spleen of a 54-year-old man with primary systemic amyloidosis could be assigned to subgroup IV of the lambda variable-region sequence. About 50% of the protein was found to be truncated in the N-terminus and lacked the first six amino acid residues. The polypeptides consisted of about 146 amino acid residues and contained traces of carbohydrate. An acceptor site for N-glycosylation was found in positions 90-93, but no glycopeptide could be isolated. Comparison of the amino acid sequence of AL protein GIL with that of the only Bence-Jones protein of subgroup IV previously studied revealed a sequence homology of 89%. A similar comparison made with other AL proteins gave sequence homologies below 66%.

scholarly journals Amino Acid Tandem Repeats within a Late Viral Gene Define the Central Variable Region of African Swine Fever Virus

Virology ◽  
1996 ◽  
Vol 220 (1) ◽  
pp. 20-27 ◽  
Author(s):  
P.M. IRUSTA ◽  
M.V. BORCA ◽  
G.F. KUTISH ◽  
Z. LU ◽  
E. CALER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tandem Repeats ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Variable Region ◽  
Fever Virus ◽  
Viral Gene ◽  
Central Variable Region

Monospecificity of the antibodies to bovine αs1-casein fragment 140–149: application to the detection of bovine milk in caprine dairy products

1995 ◽  
Vol 62 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Marie-Paule Rolland ◽  
Lotfi Bitri ◽  
Pierre Besançon
Keyword(s):  
Amino Acid ◽  
Glutamic Acid ◽  
Dairy Products ◽  
Polyclonal Antibodies ◽  
Bovine Milk ◽  
Amino Acid Residues ◽  
Native Protein ◽  
Glutamic Acid Residue ◽  
Antigen Complex ◽  
Single Difference

SUMMARYComparison of the primary sequences of bovine, ovine and caprine αs1-casein shows a deletion of eight amino acid residues in the ovine casein region 141–148, which is identical in the bovine and caprine proteins except for a single difference in position 148 (Q or E). Polyclonal antibodies raised in rabbits against the bovine casein sequence 140–149 (QELAYFYPEL) appeared monospecific for bovine αsl-casein, since no antibody-antigen complex was formed with homologous ovine or caprine proteins. These antibodies remained unable to recognize the caprine sequence in the native protein even after extensive tryptic proteolysis. The lack of immunoreactivity of the antibodies against synthetic caprine αsl-casein peptide 138–149 (VNQELAYFYPQL) suggested that the glutamic acid residue in position 148 is essential for the antigenic character of the bovine peptide. From these observations, the use of these antibodies for the detection and quantitation of bovine milk present in ovine dairy products could be extended to caprine products.

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