Biomedical subjects

Gui-Fang Shen

Publications and source records attributed to Gui-Fang Shen.

9 recordsLinked to original sources

PubMed2006

Stable expression of foot-and-mouth disease virus protein VP1 fused with cholera toxin B subunit in the potato (Solanum tuberosum).

The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity.

Blotting, Southern↗

PubMed2006

Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts.

The expression of classical swine fever virus (CSFV) structural protein E2 in different vectors, which has been shown to carry critical epitopes, has been established. Here, we reported a Chlamydomonas reinhardtii chloroplast expression vector, P64E2, containing classical swine fever virus structural protein E2 gene, which was constructed and transferred to C. reinhardtii by biolistic bombardment method. The transformants were identified by PCR, Southern blotting, Western blotting after selecting on resistant media. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.5-2% of the total soluble protein. The results of the study on the immunological activity indicated that the protein E2 expressed in C. reinhardtii chloroplasts could elicit animal bodies to produce antibodies against protein E2.

Animals↗

PubMed2006

Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii to ultraviolet B exposure.

Solar ultraviolet (UV) radiation has a great influence on green organisms, especially plankton like Chlamydomonas. A human metallothionein-2 gene, which is generally considered to have an anti-radiation function by its coding product, was transferred into the chloroplast genome of Chlamydomonas reinhardtii. To dynamically measure the UV effects on Chlamydomonas cells grown in liquid tris-acetate-phosphate medium, a new method was developed based on the relationship between the chlorophyll content of an algal culture and its absorbance at 570 nm after the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this experiment, both the wild-type and the transplastomic C. reinhardtii cells were cultivated in 96-well microplates containing liquid tris-acetate-phosphate medium in the absence or presence of zinc, copper, cadmium and cysteine. The transgenic C. reinhardtii showed a higher resistance than wild-type to UV-B exposure under all the examined conditions. Metals in the medium had positive impacts on both types of cells, but had significant influence only on the transplastomic cells. However, the high cell viability of the transgenic alga at the end of the 8 h UV-B treatment disappeared after a 20-h recovery culture. Cysteine did not protect cells from UV-B damage, but clearly enhanced the growth of both wild-type and transgenic C. reinhardtii.

Animals↗

PubMed2006

[Expression of the gene coding for a thermostable alpha-amylase from Pyrococcus furious in Chiamydomonas reinhardtii chloroplast ].

Thermostable alpha-amylase from Pyrococcus furious is an important industrial enzyme in brewing and alcohol production. Eexpression of the thermostable a-amylase in plants can reduce greatly costs in the production of alcohol using crop plants. A chloroplast expression vector, p64A, containing the thermostable alpha-amylase gene from Pyrococcus furious, was constructed with clpP-trnL-petB-chlL-rp123-rpl2 as Chlamydomonas reinhardtii plastid homologous recombinant fragments and spetinomycin-resistant aadA gene as select marker. The plasmid p64A was transferred into the chloroplast genome of C. reinhardtii by the biolistic method. Nine independently transformed lines were obtained by 100 mg/L spectinomycin selection. PCR amplification, Southern blot analysis of the transgene and cultivation in the dark all showed that the a-amylase gene had been integrated into chloroplast genome of C. reinhardtii. The activity of amylase expressed in the chloroplast of C. reinhardtii was detected by amylase activity assay and found to be as high as 77.5 u/g fresh weight of cells. These experimental results demonstrated the possibility of using transgenic chloroplasts of plant as bioreactors for production of industrial enzymes.

Chlamydomonas reinhardtii↗

PubMed2005

The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii.

The entire coding region of chlL, an essential chloroplast gene required for chlorophyll biosynthesis in the dark in Chlamydomonas reinhardtii, was precisely replaced by either the Klebsiella pneumoniae nifH (encoding the structural component of nitrogenase Fe protein) or the Escherichia coli uidA reporter gene encoding beta-glucuronidase. Homoplasmic nifH or uidA transformants were identified by Southern blots after selection on minimal medium plates for several generations. All the uidA transformants had the "yellow-in-the-dark" phenotype characteristic of chlL mutants, whereas homoplasmic nifH transformants exhibited a partial "green-in-the-dark" phenotype. NifH protein was detected in the nifH transformants but not in the wild-type strain by Western blotting. Fluorescence emission measurements also showed the existence of chlorophyll in the dark-grown nifH transformants, but not in the dark-grown uidA transformants. The nifH transplastomic form of C. reinhardtii that lacks the chlL gene can still produce chlorophyll in the dark, suggesting that the nifH product can at least partially substitute for the function of the putative "chlorophyll iron protein" encoded by chlL. Thus, introducing nitrogen fixation gene directly into a chloroplast genome is likely to be feasible and providing a possible way of engineering chloroplasts with functional nitrogenase. Notably, to introduce foreign genes without also introducing selective marker genes, a novel two-step chloroplast transformation strategy has been developed.

Animals↗

PubMed2004

[Protein splicing and its application in protein engineering].

Protein splicing, which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.

Exteins↗

PubMed2003

Chloroplast transformation in oilseed rape.

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.

Anti-Bacterial Agents↗

PubMed2002

[The insect resistance of OC and Bt transplastomic plant and the phenotype of their progenies].

The Bt gene and OC gene were cotransformed to tobacco chloroplast with particle bombardment method and spectinomycin resistance tobacco seedlings were obtained. Bioassays showed that the transgenic tobacco containing both genes had enhanced toxicity to the larvae of cotton bollworm (helicoverpa zea) by comparison with the plants containing only Bt or OC gene. Southern-blotting analysis and genetic analysis of progenies showed that the Bt and OC gene expressed and was inherited maternally to the progenies.

English Abstract↗

PubMed2000

Transplastomic Plants Homoplasmic for Foreign Transgenes.

Scientists pay more and more attention to the research on plastid engineering for its following advantages foreign genes can be integrated site-specifically into the plastid genome (plastome) there are no position effects as experienced with random insertion of transgenes in nuclear transformation pollen-mediated dispersion of transgenes can be avoided because chloroplasts are maternally transmitted gene silencing does not occur in plastids and therefore transgene expression is stable in progeny of transplastomic plants and the high ploidy level of the plastome in leaf cells makes high levels of transgene expression feasible. At the same time, however, the highly polyploid plastome makes it difficult to get transplastomic plants homoplasmic for foreign transgenes. In this work, chloroplast transforming vector pTRCH205, which carries two psbA5'-nifH-psbA3'and Prrn-aadA-TpsbA cassettes flanked by plastid DNA sequence to target their insertion between psbA and trnK operons, was constructed. Plastid transformation of Nicotiana tabacum was carried out by the biolistic delivery of transforming plasmid pTRCH205 DNA into leaf cells. Integration of nifH and aadA by two homologous recombination events via the flanking ptDNA sequences, and selective amplification of the transplastomes on MS medium with high concentration of spectinomycin, yielded resistant cell lines. All the independent transplastomic lines were subjected to three additional rounds of regeneration and were subcultured for 6--10 times through stem sections on MS medium containing 500 mg/L spectinomycin, to obtain homoplasmic tissues. The results of PCR assay and Southern blot hybridization, probed with 0.9 kb BglII/SnaBI homologous fragment, indicated that foreign genes had been integrated into the plastomes of transgenic plants, which finally became homoplasmic for foreign transgenes.

Journal Article↗