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1

Prokaryotic expression, purification of chicken calpastatin protein and production of calpastatin polyclonal antibody

100%
Ye M. H., Chen J. L., Zhao G. P., Zheng M. Q., Wen J.
Animal Science Papers and Reports
|
2013
|
tom 31
|
nr 1
The open reading frame of chicken calpastatin (CAST) gene composed of 2,301 base pairs was ligated into a prokaryotic expression vector pET21a (+) to yield pET21a - CAST. The C-terminal His-tagged CAST protein was then expressed in E. coli. BL21 (DE3). SDS-PAGE analysis confirmed the successful expression of the fusion protein following induction with isopropyl-β-Dthiogalactopyranoside (IPTG). The recombinant protein consisted of 776 amino acid residues with an apparent molecular weight of approximately 110 kDa. It was primarily expressed as a soluble protein with a heat-stable feature. After being purified by Ni2+-NTA affinity resin, a polyclonal antibody was raised against the purified His-tagged CAST protein in rabbits. The reactivity and specificity of the polyclonal antibody were both subsequently characterized by ELISA. The study provides an important experimental tool for further research on the quantification of chicken CAST protein.
2

Sensitivity and specificity of high-resolution melting analysis in screening unknown SNPs and genotyping a known mutation

100%
Ye M. H., Chen J. L., Zhao G. P., Zheng M. Q., Wen J.
Animal Science Papers and Reports
|
2010
|
tom 28
|
nr 2
High-resolution melting analysis (HRMA) was used to screen potential SNPs in the exons of chicken CAPN1 (μ-calpain/large subunit) gene. A total of 312 DNA samples from Beijing-you chickens were used for detection. Twelve pairs of primer were designed to amplify twelve different exons and SNPs were detected in five of them. HRMA was also compared with PCR-SSCP analysis for genotyping of a known SNP site in the chicken adipocyte fatty acid binding protein gene (A-FABP). Amplicons of 275-bp fragment, bracketing the polymorphic site, were grouped by PCR-SSCP into three genotypem designated as CC, TT and CT. Small amplicons (56 bp) within the 275-bp fragments were designed to maximize the Tm difference between homozygotes and to genotype all possible three genotypes after a single melting analysis successfully. Results from different methods were cross-validated and sequencing results from randomly selected heterozygotes and homozygotes confirmed the specificity of HRM technique. The full consistency proved that HRMA was a useful tool for rapid, close-tube genotyping of polymorphic sites. It has great potential for SNPs detection and scanning especially on a large scale.
3

Screening of differentially expressed genes induced by water-soluble extracts from pollen during honeybee caste determination

100%
Zhou B., Ye M. H., Zhang K., Wu Y. W., Ding J. T.
Journal of Animal and Feed Sciences
|
2010
|
tom 19
|
nr 2
292-306
In order to screen differentially expressed genes during caste differentiation induced by pollen, larvae of Apis mellifer ligustica were developed in an incubator under controlled temperature and relative humidity until emergence. Worker bee morphology was induced by addition of water-soluble extracts from pollen in larval diets. After all of the developmental periods, the external characters and developmental degree of ovaries were evaluated to confirm the caste of new adults. Transcripts from larvae of developing queens and workers were profiled by differential-display RT-PCR analysis and differentially expressed fragments were reconfirmed by dot-blot assay. Our results showed that caste differentiation was triggered by the intake of pollen extracts. Six transcript-derived fragments were isolated and three of them are reported for the first time. We conclude that differential display is a feasible approach to identifying differentially expressed genes. The identification of up-regulated caste-differentiation-related genes provided interesting clues about the activation of biochemical steps relevant to this progress.
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