Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T. sergenti (Korea isolate).The previously recognized major bands, 18 kDa, 29 kDa, 34 kDa, and 45 kDa, were excised after electrophoresis and trasferred to PVDF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antiginic site) for antigen determinants of the 45 kDa,34 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypeptide showed no reaction with anti-T. sergenti hyperimmune serum.
parasitology-protozoa ; Theileria sergenti ; amino acid sequence ; synthetic peptide ; predicted antigenic value ; amino acid

OBJECTIVETo develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).

METHODSTo allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.

RESULTSThree 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.

CONCLUSIONPCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.

Base Sequence ; Cloning, Molecular ; Genes, Synthetic ; genetics ; Lactococcus ; genetics ; Peptide Fragments ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; metabolism ; Tetanus Toxin ; genetics ; metabolism

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Enzyme-Linked Immunosorbent Assay ; Genes, Synthetic ; genetics ; physiology ; Models, Theoretical ; Peptide Library ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transcription Factors ; chemical synthesis ; chemistry ; metabolism ; Zinc Fingers ; genetics ; physiology

At present time, the widely used hepatitis B virus( HBV) vaccines consist of only the small hepatitis B surface antigen expressed in yeast or CHO cells. The frequency of non-responders to these vaccines has increased the demand for a more immunogenic vaccine. Some studies have suggested that the addition of preS region to the vaccine will improve its efficacy. However, the large protein (L) containing the whole preS region can not be effectively expressed in vitro. To overcome this problem, two chimeric contructs, SS1, surface gene containing preS1 region at C-terminus and SS2, surface gene containing preS2 region at C-terminus, were constructed and effectively expressed in our previous studies. Here we further constructed an expression vector containing both SS1 and SS2 expression cassettes by separation and ligation the SS2 cassette to a linearized SS1 expression vector pAO815-SS1. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation. A high-level expression strain (GS115-SS1S2) was established by primary screening for His+ transformants and further analysis for induction products. ELISA results demonstrated that the expressed protein had S, preS1 and preS2 antigenicities simultaneously. Western blotting showed that the product can bind to all of the three antibodies, anti-S, anti-preS1 and anti-preS2. The expression protein was present in the form of particles of 20-35 nm diameter and the yield of recombinant particles reached 300-600 mg/L by fermentation. The SS1 and SS2 polypeptides kept intact in purified particles, suggesting that the stability of preS region has been significantly improved.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Epitopes ; genetics ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Peptide Fragments ; genetics ; immunology ; Pichia ; genetics ; Protein Precursors ; genetics ; immunology ; Vaccines, Synthetic ; immunology

Even though chemistry is now in place that potentially allows high coupling efficiencies to be attained, successful coupling is usually a challenge when so-called "difficult sequences" is encountered in peptide synthesis. Some factors that affect the coupling efficiency have been discussed and related methods to overcome those obstacles have been introduced in present review. All suggestions proposed here are valuable and also feasible to improve the coupling completeness in both liquid-phase or solid-phase
Acylation ; Amino Acid Sequence ; Molecular Structure ; Organophosphorus Compounds ; chemistry ; Peptide Fragments ; chemical synthesis ; chemistry ; Peptides ; chemical synthesis ; chemistry ; Proline ; analogs & derivatives ; chemistry ; Resins, Synthetic ; chemistry ; Temperature ; Thiazoles ; chemistry ; Thiocyanates ; chemistry ; Triazoles ; chemistry

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to A1a and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to > 95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1 x 10(3) 50% lethal doses (LD50s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.
Escherichia coli ; genetics ; metabolism ; Mutant Proteins ; biosynthesis ; genetics ; immunology ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Protein Conformation ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Tetanus ; prevention & control ; Tetanus Toxin ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

To improve the efficacy of peptide P277 in preventing autoimmune diabetes, heat shock protein 65 kD (HSP65) of Mycobacterium tuberculosis var. bovis was fused with linear polypeptide epitope of P277 and expressed as soluble protein in Escherichia coli. The fusion protein HSP65-P277 was purified by anion exchange column chromatography and then used to immunize prediabetic NOD mice with three ip inoculations in absence of adjuvants. Serum samples from the immunized mice were collected monthly and the concentration of blood glucose was measured. The study showed that administration of HSP65-P277 to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself or HSP65. Fused to heat shock protein 65 of Mycobacterium tuberculosis could improve the efficacy of diabetes prevention of P277 in nonobese diabetic mice. The results suggest the fusion protein of HSP65-P277 would be useful for treating insulin-dependent diabetes mellitus.
Animals ; Bacterial Proteins ; biosynthesis ; genetics ; immunology ; Chaperonin 60 ; Chaperonins ; biosynthesis ; genetics ; immunology ; Diabetes Mellitus, Type 1 ; prevention & control ; Escherichia coli ; genetics ; metabolism ; Female ; Heat-Shock Proteins ; genetics ; immunology ; Immunization ; Mice ; Mice, Inbred NOD ; Mycobacterium bovis ; Peptide Fragments ; biosynthesis ; genetics ; immunology ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Synthetic ; genetics ; immunology

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, commonly known as St. John's wort. Hypericin has attracted a growing attention of the pharmaceutical industry because of its potential application to various therapies, including the treatment of depression and remarkable antiviral and photodynamic activities, hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which, however, has not formed common opinion so far. Six pairs of primers specific to hyp-1 gene were synthesized. The rapid cloning of hyp-1 gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pET32ahyp containing hyp-1 gene was constructed and was transformed into E. coli to induce heterologous expression. SDS-PAGE and Western blot results showed the recombinant Hyp-1 protein was expressed successfully in E. coli. The soluble fraction was used to test the function of the recombinant Hyp-1. Hypericin was detected by LC-MS/MS with emodin as a substrate under in vitro conditions. The above results corroborated the Hyp-1 function, a confusing question, which lay a material foundation for the synthesis of hypericin by synthetic biotechnology.
Antidepressive Agents ; isolation & purification ; metabolism ; Antiviral Agents ; isolation & purification ; metabolism ; Chemistry Techniques, Synthetic ; Emodin ; metabolism ; Escherichia coli ; metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; Genetic Vectors ; Hypericum ; chemistry ; Peptide Synthases ; genetics ; isolation & purification ; metabolism ; Perylene ; analogs & derivatives ; isolation & purification ; metabolism ; Plant Proteins ; genetics ; isolation & purification ; metabolism ; Plants, Medicinal ; chemistry ; Recombinant Proteins ; genetics ; metabolism ; Transformation, Genetic