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Cloning and Expression of Gene Coding Cathepsin L of Rhipicephalus annulatus 全文
2020
Sattari Tabrizi, Saeed | Nabian, Sedigheh | Ebrahimzadeh, Elahe | Shayan, Parviz | Alidadi, Naser | Amininia, Narges
BACKGROUND: Ticks are one of the most important ectoparasites in animals that cause economic losses in livestock industry. So, removal or reduction of ticks on animals is necessary. Cysteine proteases are among the compounds that play an important role in the physiological action of ticks and are a good candidate for the anti-tick vaccine. Cathepsins is one of the most important cysteine proteases. OBJECTIVES: The aim of this study was cloning and expression of recombinant cathepsin L gene of Rhipicephalus annulatus in order to evaluate its immunogenicity. METHODS: After collection the ticks were cultivated. Then RNA was extracted from ticks, cDNA was synthesized by using specific primer of cathepsin and amplification by RT-PCR. The desired genes were cloned into expressional pQE30 plasmid. Further, a shorter sequence of the cathepsin gene (654 bp) was prepared as a synthetic plasmid. The expression of the protein produced by both recombinant plasmids in the E.coliBL21 prokaryotic expression system is carried out and the immunity of the recombinant proteins was evaluated by Dot Blot and Western Blot using serum of challenged rabbits with recombinant protein and calves infected with ticks were examined and compared. RESULTS: The results of this study indicate that the protein derived from recombinant plasmid No. 2 had higher expression and purity due to its solubility. Also, the challenge of rabbit serum with these proteins was able to identify both recombinant proteins. But the serum of challenged calves with ticks did not show a satisfactory response with recombinant proteins. CONCLUSIONS: Although the sera reaction of calves infested with ticks was lower than the challenged rabbits sera with cathepsin L, this result was expected, because L cathepsin protein is considered as a concealed antigen. Overall, the recombinant cathepsin L could be an appropriate candidate for immunizing calves against Rhipicephalus annulatus, although it seems further investigations are necessary.
显示更多 [+] 显示较少 [-]Production of Recombinant FanC of Enterotoxigenic Escherichia Coli Associated with Calf Diarrhea 全文
2018
Tabatabaei, Saeid | Nikbakhat Brujeni, Gholamraza | Tebyanian, Majid | Zainel, Khalil | Jalali, Seyed Amir Hossein
BACKGROUND: Diarrhea is a common disease in the neonate calf which imposes significant economic burden on cattle industry around the world. During the first week after birth, Enterotoxigenic E.coli (ETEC) strains carrying F5 fimbria are one of the most important pathogens causing calf diarrhea. F5 fimbria is involved in early stage of pathogenesis and is responsible for attachment of bacteria to enterocytes; this attachment is mediated by FanC protein of F5 fimbria. Antibodies directed against F5 fimbriae play a significant role in prevention and control of the disease. Objectives: Evaluation and expression of recombinant expression of F5 Fimbriae of Enterotoxigenic Escherichia Coli associated with calf diarrhea. Methods: In the present study, the fanC region of F5 fimbria was cloned in a pET28a plasmid. Results: The recombinant construct was confirmed by sequencing and protein production in Escherichia coli BL21 (DE3) was evaluated by western blotting procedure. Conclusions: Based on our findings, the recombinant FanC protein or the BL21 (DE3) strain are suitable candidates to develop an effective vaccine against calf colibacillosis or use in a diagnostic kit for F5+ ETEC.
显示更多 [+] 显示较少 [-]Production of the phospholipase D and heat-shock protein (HSP)-60 recombinant proteins from Corynebacterium pseudotuberculosis 全文
2017
Boroon, Fatemeh | Seyfi Abad Shapouri, Masoud Reza | ghorbanpoor, masood | Gharibi, Dariush | Esmaeelzadeh, Saleh
BACKGROUND: Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, is one of the most important diseases of sheep and goats, causing considerable losses for herd owners. Phospholipase D (PLD) is a potent exotoxin produced by C. pseudotuberculosis and it has been considered as the major virulence factor for this bacterium, possibly contributing to the spread of the bacteria from the initial site of infection to secondary sites within the host. Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. OBJECTIVES: The aim of this study was the cloning and expression of the PLD and HSP60 genes of C. pseudotuberculosis, used subsequently to evaluate the protectivity of these recombinant proteins for vaccine development against this bacterium. METHODS: PLD and HSP60 genes were cloned into pMAL-c2X vector and recombinant plasmids construct was transformed to DH5 strain of E. coli. Expression of the proteins was shown by SDS-PAGE and accuracy of the cloned genes was confirmed by nucleotide sequence analysis. RESULTS: The transformed E. coli strain DH5 expressed PLD and HSP60 proteins effectively. The expressed fusion protein was found almost entirely in the soluble form. CONCLUSIONS: In the following studies the immunogenicity and protectivity of these recombinant proteins against C. pseudotuberculosis infections can be assessed.
显示更多 [+] 显示较少 [-]Cloning and differential expression analyses of Cdc42 from sheep 全文
2018
Yang Yong-Jie | Liu Zeng-Shan | Lu Shi-Ying | Hu Pan | Li Chuang | Ahmad Waqas | Li Yan-Song | Xu Yun-Ming | Tang Feng | Zhou Yu | Ren Hong-Lin
Cloning and differential expression analyses of Cdc42 from sheep 全文
2018
Yang Yong-Jie | Liu Zeng-Shan | Lu Shi-Ying | Hu Pan | Li Chuang | Ahmad Waqas | Li Yan-Song | Xu Yun-Ming | Tang Feng | Zhou Yu | Ren Hong-Lin
Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis.
显示更多 [+] 显示较少 [-]Cloning and differential expression analyses of Cdc42 from sheep 全文
2018
Yang, Yong-Jie | Liu, Zeng-Shan | Lu, Shi-Ying | Hu, Pan | Li, Chuang | Ahmad, Waqas | Li, Yan-Song | Xu, Yun-Ming | Tang, Feng | Zhou, Yu | Ren, Hong-Lin
Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis. Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR. The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity. OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.
显示更多 [+] 显示较少 [-]MOLECULAR CLONING AND SEQUENCE ANALYSIS OF TES-120 GENE OF Toxocara canis EXCRETORY-SECRETORY ANTIGEN 全文
2022
K.T. Kavitha | C. Sreekumar | Bhaskaran Ravi Latha | A. Mangala Gowri
The gene encoding the excretory-secretory antigen of TES-120 from larvae of Toxocara canis was cloned and the sequence was analyzed for utilization as a diagnostic molecule. The total RNA was isolated from T. canis infective larvae and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Amplification by PCR was performed with cDNA as a template and specific primers of the gene of TES-120, yielding an amplicon size of 530 bp. The amplicon was cloned into pRSETa expression vector. The recombinant clones were transformed into BL21 (DE3) pLysS E. coli expression host and the clone was confirmed by colony PCR, restriction enzyme analysis and sequencing. The nucleotide sequencing of TES-120 gene of TN isolate showed 99.8 % homology with the previously published sequences of UK (U39815.1) and Venezuela isolate (KU951901.1) and 97.0 % homology with the published sequences of Iraq isolate (LC328969.1). Phylogenetic tree revealed close relationship of TN isolate with Venezuela isolate, which had a common ancestor with the UK isolate and it is distantly related to Malaysia isolate of T. cati (KP71707).
显示更多 [+] 显示较少 [-]Development of a DNA probe for identification of bovine herpesvirus 4
1993
Galik, P.K. | Van Santen, V.L. | Stringfellow, D.A. | Bird, R.C. | Wright, J.C. | Smith, P.C.
A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 microgram of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.
显示更多 [+] 显示较少 [-]Detection of bovine viral diarrhea virus, using degenerate oligonucleotide primers and the polymerase chain reaction
1991
Ward, P. | Misra, V.
A technique for detection of bovine viral diarrhea virus (BVDV) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of BVDV and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral RNA in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of BVDV by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.
显示更多 [+] 显示较少 [-]Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus
1991
Elton, D.M. | Bonass, W.A. | Killington, R.A. | Meredith, D.M. | Halliburton, I.W.
The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.
显示更多 [+] 显示较少 [-]Sequence and phylogenetic analysis of chicken anaemia virus obtained from backyard and commercial chickens in Nigeria 全文
2008
Oluwayelu, D.O.(University of Ibadan Department of Veterinary Microbiology and Parasitology) | Todd, D.(Veterinary Sciences Division Department of Agriculture and Rural Development for Northern Ireland) | Olaleye, O.D.(University of Ibadan College of Medicine Department of Virology)
This work reports the first molecular analysis study of chicken anaemia virus (CAV) in backyard chickens in Africa using molecular cloning and sequence analysis to characterize CAV strains obtained from commercial chickens and Nigerian backyard chickens. Partial VP1 gene sequences were determined for three CAVs from commercial chickens and for six CAV variants present in samples from a backyard chicken. Multiple alignment analysis revealed that the 6 % and 4 % nucleotide diversity obtained respectively for the commercial and backyard chicken strains translated to only 2 % amino acid diversity for each breed. Overall, the amino acid composition of Nigerian CAVs was found to be highly conserved. Since the partial VP1 gene sequence of two backyard chicken cloned CAV strains (NGR/Cl-8 and NGR/Cl-9) were almost identical and evolutionarily closely related to the commercial chicken strains NGR-1, and NGR-4 and NGR-5, respectively, we concluded that CAV infections had crossed the farm boundary.
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