EXPRESSION CLONING OF A PROTECTIVE LEISHMANIA ANTIGEN

Parasite-specific CD8+ T cells have been shown to transfer protection against nLeishmania major in susceptible BALB/c mice.An epitope-tagged expression library was used to identify the antigen recognized by a protective CD8+ T cells clone. The expression library allowed recombinant proteins made in bacteria to be captured by macrophages for presentation to T cells restricted to major histompatibility complex class n. A conserved 36-kilodalton member of the tryptophanaspartic acid repeat family of proteins was identified that was expressed in both stages of the parasite life cycle. A 24-kilodalton portion of this antigen protected susceptible mice when administered as a vaccine with interleukin-12before injection.

Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and Cloning and Expression of Betaine Aldehyde Dehydrogenase(BADH) Gene

The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.2.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.

Cloning and heterologous expression of pro-2127,a gene encoding cold-active protease from Pseudoalteromonas sp.QI-1

The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.

Porcine methionine sulfoxide reductase B3:molecular cloning,tissue-specific expression profiles,and polymorphisms associated with ear size in Sus scrofa

Background： In Sus scrofa, methionine sulfoxide reductase B3（MSRB3） is a crucial candidate gene for ear size, and an important conformational trait of pig breeds. However, challenges in MSRB3 c DNA amplification have prevented further identification of MSRB3 allelic variants influencing pig ear size.Results： We cloned a full-length c DNA sequence of porcine MSRB3 by rapid-amplification of c DNA ends. The3,765-bp gene contained a 5＇-untranslated region（UTR）（190 bp）, a coding region（552 bp）, and a 3＇-UTR（3,016 bp） and shared 84 %, 84 %, 87 %, 86 %, and 70 % sequence identities with human, orangutan, mouse, chicken, and zebrafish,respectively. The gene encoded a 183-amino acid protein, which shared 88 %, 91 %, 89 %, 86 %, and 67 % identities with human, orangutan, mouse, chicken, and zebrafish, respectively. Tissue expression analysis using q RT-PCR revealed that MSRB3 was expressed in the heart, liver, lung, kidney, spleen, ear, muscle, fat, lymph, skeletal, and hypothalamic tissues. Three single nucleotide polymorphisms（SNPs） were identified in MSRB3： c.-735 C 〉 T in the 5＇ flanking region,c.2571 T 〉 C in the 3＇-UTR, and a synonymous mutation of c.484 T 〉 C in the coding region. The SNPs c.-735 C 〉 T and c.2571 T 〉 C were significantly associated with ear size in a Large White × Minzhu F2 population other than in Beijing Black pigs. Subsequently, at SNP c.-735 C 〉 T, the m RNA of MSRB3 was significantly higher expressed in ears of individuals with the TT genotype（Minzhu） than those with CC（Large White）.Conclusions： The porcine MSRB3 owned a 3,765-bp full-length c DNA sequence and was detected to express in ear tissue. Two SNPs of this gene were shown to be significantly associated with ear size in a Large White × Minzhu intercross population instead of Beijing Black pig population. What＇s more, the individuals with higher m RNA expression of MSRB3 have larger ear sizes. These results provide useful information for further functional analyses of MSRB3 influencing ear size in pigs.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Cloning and Expression Analysis of Allene Oxide Synthase Gene Ls AOS in Lilium‘Siberia'

[Objectives] Allene oxide synthase（ AOS）,the first specific enzyme in the biosynthetic pathway of jasmonate（ JA）,has an important role in regulating JA accumulation in plants. This study was conducted to amplify full-length AOS gene from Lilium‘Siberia＇（ LsAOS） and to explore the spatiotemporal expression of this gene. [Methods]LsAOS was cloned by rapid amplification of c DNA ends（ RACE） and analyzed bioinformatically. LsAOS expression level in nine organs/tissues and at four flowering stages was determined by quantitative PCR（ q-PCR）. [Results] LsAOS gene has an open reading frame of 1 542 bp,encoding a protein of 514 amino acids. The LsAOS protein sequence was more homologous with the AOS protein of Musa acuminata Colla than that in any other plant species.The LsAOS expression reached the maximum level at bud stage,and then gradually decreased over time during the flowering period. The LsAOS expression level in leaves was the highest,followed by in inner petals,roots,stems,etc. [Conclusions]JA participates in the whole process of plant development,and is closely related to stress resistance and secondary metabolism. AOS is one of the key genes in JA pathway and plays an important role in regulating the biosynthesis of JA.

Molecular Cloning,Expression,and Characterization of an Adenylyl Cyclase-associated Protein from Gossypium arboreum Fuzzless Mutant

CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild

Molecular Cloning and Expression Analysis of a Cys2/His2 Type Zinc Finger Protein Gene in Upland Cotton

The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

Construction of recombinant plasmid and prokaryotic expression in E. Coli and biological activity analysis of human placenta arresten gene

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.

Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera:Thripidae)

Heat shock protein 70（HSP70） is one of the most important members in the heat shock protein family, and plays important roles in the thermotolerance of insect. To explore the molecular mechanism of thermotolerance of Frankliniella occidentalis adults, the difference in the expression of HSP70s in F. occidentalis male or female adults under the thermal stress was studied under the laboratory conditions. Two full length c DNAs of HSP70s gene（Fohsc704 and Fohsc705） were cloned from F. occidentalis by using RT-PCR and RACE. The genomic sequence was demonstrated by genomic validation, and the position and size of the intron were analyzed by sequence analysis of c DNA. Real-time PCR was used to analyze the HSP70 expression patterns. The c DNA of Fohsc704 and Fohsc705 possessed 2 073 and 1 476 bp which encoded 690 and 491 amino acids（aa） with a calculated molecular weight of 75 and 54 k Da, respectively. Four introns in Fohsc704 and six introns in Fohsc705 protein were found. However, the HSP70 protein sequences in our study were ended with EKKN and GIFL, which were different from the reported Fo HSP70s. Various expression patterns of Fohsc704 and Fohsc705 were found in both genders of F. occidentalis under thermal stress. The expression of Fohsc704 and Fohsc705 reached to the highest level at –12 and –8°C in male adults, respectively, and Fohsc705 expressed the highest level at 33°C in female adults. In conclusion, HSP70s of F. occidentalis in our study are novel heat shock proteins. There were difference in expression patterns of the two hsc70s in genders of F. occidentalis, and the two HSP70s play important roles in the thermotolerance of F. occidentalis.

Cloning,Tissue Distribution,and Transmembrane Orientation of the Olfactory Co-Receptor Orco from Two Important Lepidopteran Rice Pests,the Leaffolder(Cnaphalocrocis medinalis) and the Striped Stem Borer(Chilo suppressalis)

In insects,the sense of smell is mainly mediated by olfactory receptors（Ors）.Olfactory co-receptor（Orco）,which is coexpressed with the Ors in almost all olfactory receptor neurons（ORNs）,is demonstrated to be an essential component in the insect olfactory system.It can be potential target for developing novel olfactory-disruption strategy to control insect pests.In this study,two full-length cDNA sequences encoding Orcos（CmedOrco and ChsupOrco） were cloned from two Lepidopteran rice pests,the rice leaffolder,Cnaphalocrocis medinalis and the rice striped stem borer,Chilo suppressalis.The amino acid sequences of CmedOrco and ChsupOrco showed high similarity to the previously identified Orcos from other insect species. Bioinformatic prediction and cellular immunofluorescence indicated that CmedOrco and ChsupOrco were both seven-transmembrane proteins with intracellular N-termini and extracellular C-termini.mRNA expression levels of the two Orcos were much higher in male and female antennae than those in non-olfactory tissues,and the ChsupOrco transcripts reached a peak level in adults compared to other life stages.Our results provide a foundation from which it will be possible to elucidate the roles of Orco in moth olfaction and for the development of environment-friendly management strategies of these two rice insect pests.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Cloning and expression analysis in mature individuals of two chicken type-Ⅱ GnRH (cGnRH-Ⅱ) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide fam-ily, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II pre-cursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplica-tion hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the ex-pression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Cloning of Chinese obese cDNA and its expression in E coli

Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology

Identification and Cloning of Differentially Expressed Genes Involved in the Interaction Between Potato and Phytophthora infestans using a Subtractive Hybridization and cDNA-AFLP Combinational Approach

Using a subtractive hybridization （SH）/cDNA-AFLP combinational approach, differentially expressed genes involved in the potato-Phytophthora infestans interaction were identified. These included genes potentially controlling pathogenesis or avr genes in P. infestans as well as those potentially involved in potato resistance or susceptibility to this pathogen. Forty-one differentially expressed transcript, derived fragments （TDFs）, resulting from the interaction, were cloned and sequenced. Two TDFs, suggested as potential pathogenicity factors, have sequence similarity to N-succinyl diaminopimelate aminotransferase and a transcriptional regulator, TetR family gene, respectively. Two other TDFs, suggested as potential avr genes, have sequence similarity to an EST sequence from Avr41Cf.41Avr91Cf- 9 and a P. infestans avirulence-associated gene, respectively. Genes＇ expression and origin were confirmed using Southern blots, Northern blots and qRT-PCR, he., potential resistance gene DL81 was induced at 12 hpi in the moderately resistant cultivar, whereas it was down-regulated as early as 6 hpi in the susceptible cultivar. On the other hand, DL21 was induced at 6 hpi （3.38-fold） in response to the highly aggressive isolate （US8） and strongly up-regulated thereafter （25.13-fold at 120 hpi.）, whereas it was only slightly up-regulated in response to the weakly aggressive isolate US11 （3.82-fold at 96 hpi）, suggesting its potential involvement as a susceptibility gene.