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1.
Cloning and expression of swine myostatin gene and its application in animal immunization trial 总被引:7,自引:0,他引:7
MA Xianyong CAO Yongchang SHU Dingming & BI Yingzuo Genetic Engineering Laboratory College of Animal Science South China Agricultural University Guangzhou China 《中国科学:生命科学英文版》2005,48(4):368-374
An important performance trait target in swine breeding is the percentage of the lean meat within the total consumable pork. China has numerous indige-nous swine breeds, but the percentage of the lean meat percentage is generally low. Thus, increasing the per-centage of the lean meat would improve the pork qual-ity, which would have great importance in the devel-opment of our livestock industry as well as the opti-mization of the consumers’ dietary. Throughout the years researchers have been … 相似文献
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Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily that functions as a negative regulator
of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function
of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5′ and 3′ flanking
sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage
domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart.
It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several
types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further,
it was demonstrated that the 5′ flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures. 相似文献
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Tae-Geum Kim Bang-Geul Kim Mi-Young Kim Jae-Kwon Choi Eun-Sun Jung Moon-Sik Yang 《Molecular biotechnology》2010,44(1):14-21
Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment.
Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The
synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated
transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis,
respectively. The binding activity of LTB protein expressed in transgenic rice callus to GM1-ganglioside, a receptor for biologically active LTB, was confirmed by GM1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody
titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to GM1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers.
Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response.
Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than
mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic
rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that
transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic
E. coli heat-labile toxin. 相似文献
8.
Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models
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Çiğdem Yılmaz Aycan Apak Erkan Özcengiz Gülay Özcengiz 《Microbiology and immunology》2016,60(11):717-724
Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN‐γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post‐challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD–MPLA than in those that had received rFeSOD‐Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD–MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific‐IFN‐γ responses were significantly stronger in the group vaccinated with rFeSOD–MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component. 相似文献
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Polymorphisms of myostatin (MSTN) gene were investigated as a candidate marker for goat growth in 687 individuals by gene sequencing and polymerase chain
reaction restriction fragment length polymorphism methods. Three potential genotypes (AA, AB and BB) of 5 bp indel (1,256
TTTTA/−) in 5′UTR were detected in four breeds. The polymorphism (CC, CD and DD) of substitution (1,388 T/A) in exon 1 was
only segregating in Boer. Genotype AB resulted in significant increases in body weights at birth (BW0), 90 (BW90) and 300
(BW300) days of age, and birth body length compared to BB (P < 0.05). Whilst genotype CD contributed to heavier BW0 and BW90, and larger birth body height (BH) compared to CC (P < 0.05). Individuals either with AB or CD genotype also had greater values in other body sizes, although no significant differences
appeared (P > 0.05). When in combination, the combined genotype ABCD displayed positive impacts on better growth traits in BW0, BW90,
BW300, BH and BCG (P < 0.05). These results indicate that these markers in myostatin (MSTN) are associated with Boer growth and may be useful for marker assisted selection. 相似文献
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Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice 总被引:4,自引:0,他引:4
Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C57BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis. 相似文献
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A promoter polymorphism of bovine Myostatin (MSTN) gene g.−371T>A was screened in Holstein and two Korean indigenous cattle breeds, Hanwoo and Jeju Black cattle (JBC). The
MSTN g.−371T>A polymorphism was found in all three cattle breeds tested. An allele MSTN g.−371A was the most frequent in the JBC breed among breeds tested. The association of MSTN genotypes for carcass traits was also tested in the Hanwoo population. Significant differences were found between the genotypes
and level of meat quality grade index which converted the marbling score levels (P < 0.05), reflecting the metabolic role of MSTN for inhibition of preadipocyte differentiation in intramuscular fat deposition. In addition, significant differences were
found for fat color index of backfat according to MSTN genotypes (P < 0.05), suggesting that MSTN may play a role in deposition of white-yellow adipocytes in backfat. However, there was no detection of significant association
of genotypes with the live weight, carcass weight, backfat thickness, eye muscle area, marbling score, or meat color index
(P > 0.05). Despite the lack of statistical association, wild type g.−371T/-showed association patterns similar to those of A/A homozygotes, such as heavier weights, thinner backfat, larger eye muscle area, and lower marbling score. The results of the
present study suggest that MSTN promoter polymorphism g.−371T>A may affect carcass traits, which could be a useful molecular marker for planning improvements
in the economic traits of Korean cattle breeds. 相似文献
12.
Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice 总被引:1,自引:0,他引:1
Perea Arango I Loza Rubio E Rojas Anaya E Olivera Flores T Gonzalez de la Vara L Gómez Lim MA 《Plant cell reports》2008,27(4):677-685
Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins
including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus
in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1–5% of total soluble protein
in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene
was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with
tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized
i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments
where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were
not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work
is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice. 相似文献
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Junjie Luo Zhiyuan Song Shengli Yu Dan Cui Benli Wang Fangrong Ding Song Li Yunping Dai Ning Li 《PloS one》2014,9(4)
Genetically engineered zinc-finger nucleases (ZFNs) are useful for marker-free gene targeting using a one-step approach. We used ZFNs to efficiently disrupt bovine myostatin (MSTN), which was identified previously as the gene responsible for double muscling in cattle. The mutation efficiency of bovine somatic cells was approximately 20%, and the biallelic mutation efficiency was 8.3%. To evaluate the function of the mutated MSTN locus before somatic cell nuclear transfer, MSTN mRNA and protein expression was examined in four mutant cell colonies. We generated marker-gene-free cloned cattle, in which the MSTN biallelic mutations consisted of a 6-bp deletion in one of the alleles and a 117-bp deletion and 9-bp insertion in the other allele, resulting in at least four distinct mRNA splice variants. In the MSTN mutant cattle, the total amount of MSTN protein with the C-terminal domain was reduced by approximately 50%, and hypertrophied muscle fibers of the quadriceps and the double-muscled phenotype appeared at one month of age. Our proof-of-concept study is the first to produce MSTN mutations in cattle, and may allow the development of genetically modified strains of double-muscled cattle. 相似文献
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根据GenBank公开序列自行设计一对引物,通过RT-PCR扩增出小鼠肝炎病毒的全长S1基因,并将其插入真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1。将重组质粒转染COS-7细胞,采用间接免疫荧光检测出S1蛋白的体外表达。将重组质粒转入减毒鼠伤寒沙门氏菌SL7207中,构建出运送DNA疫苗的重组沙门氏菌SL7207(pVAX1-S1)。分别以5×108CFU、1×109CFU、2×109CFU剂量的重组菌口服接种6周龄BALB/c小鼠,试验结果表明,重组菌对小鼠具有良好的安全性。以1×109CFU剂量的重组菌口服免疫小鼠,抗体检测结果显示,在二免后两周和三免后两周,重组菌免疫组的血清抗体水平与SL7207(pVAX1)空载体免疫组间分别存在显著性差异(P<0.05)和极显著性差异(P<0.01)。在三免后两周重组菌免疫组出现了较高水平的肠黏膜抗体。 相似文献
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The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and
ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG
positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with
the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii. 相似文献
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Seung-Moon Park Eun-Jin Choi Tae-Ho Kwon Yong-Suk Jang Han-Sang Yoo Woo Bong Choi Bong-Kyun Park Dae-Hyuk Kim 《Biotechnology and Bioprocess Engineering》2005,10(4):362-366
Actinobacillus pleuropneumoniae is an important pig pathogen, which is responsible for swine pleuropneumonia, a highly contagious respiratory infection.
To develop subunit vaccines forA. pleuropneumoniae infection, the Apx toxin genes,apxI andapxII, which are thought to be important for protective immunity, were expressed inSaccharomyces cerevisiae, and the induction of immune responses in mice was examined. TheapxI andapxII genes were placed under the control of a yeast hybridADH2-GPD promoter (AG), consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. Western blot analysis confirmed that both toxins were successfully expressed in the yeast. The ApxIA and ApxIIA-specific
IgG antibody response assays showed dose dependent increases in the antigen-specific IgG antibody titers. The challenge test
revealed that ninety percent of the mice immunized with ApxIIA or a mixture of ApxIA and ApxIIA, and sixty percent of mice
immunized with ApxIA survived, while none of those in the control groups survived longer than 36 h. These results suggest
that vaccination of the yeast expressing the ApxI and ApxII antigens is effective for the induction of protective immune responses
againstA. pleuropneumoniae infections in mice. 相似文献
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M. Crispo A. P. Mulet L. Tesson N. Barrera F. Cuadro P. C. dos Santos-Neto T. H. Nguyen A. Crénéguy L. Brusselle I. Anegón A. Menchaca 《PloS one》2015,10(8)
While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock. 相似文献
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Zhiqiang Li Shuli Wang Shujuan Wei Guangli Yang Chunmei Zhang Li Xi Jinliang Zhang Yanyan Cui Junfang Hao Huan Zhang Hui Zhang 《Microbial biotechnology》2022,15(6):1811-1823
Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis. 相似文献
19.
The Molecular Characterization and Immunity Identification of Microneme 3 of Eimeria acervulina
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ZhenChao Zhang XinChao Liu XinChao Yang LianRui Liu Shuai Wang MingMin Lu Mohammad Ehsan Javaid A. Gadahi XiaoKai Song LiXin Xu RuoFeng Yan XiangRui Li 《The Journal of eukaryotic microbiology》2016,63(6):709-721