Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody
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The high-affinity Na+/glucose contransporter Sglt1 is one of the important members of the sodium:solute symporter family(SSF),belonging to the homologous family 5(SLC5).The Sglt1 plays an important role in accumulating glucoses from intestinal or kidney epithelial cells against an adverse concentration gradient and maintaining the adjustment of metabolism.So far,few studies on Na+/glucose cotransporter have been reported in the freshwater fishes.This research would develop special antibodies of Sglt1,which could supply the foundation for the research of glucose metabolism in Cyprinus carpio intestines on molecular level.To study the molecular mechanism of glucose metabolism in freshwater fishes,the full-length cDNA of sglt1 in C.carpio with an ORF of 1 977 bp was cloned and the antigenicity of Sglt1 was first predicted.92(544-637)amino acids with strong antigenicity and immunogenicity were selected as target section of sglt1 in C.carpio and were cloned into plasmid pET-32a(+)vector which had restriction sites for EcoRⅠ and HindⅢ.The rcombinant plasmid named pET-32a(+)-sglt1-P was transformed into E.coli Rosetta.The Sglt1-P fusion protein of approximately 30 ku was highly expressed in E.coli Rosetta after being induced with IPTG.The purified fusion protein was used as antigen to immunize New Zealand Rabbits with ear margin veins by subcutaneous injection with power.The results of ELISA showed that the titer of the antiserum was about 1∶ 105.The Sglt1-P polyclonal antibody was used to determine the expression of Sglt1 protein in C.carpio intestines through immunohistochemical method.The result revealed that the antibody performed high affinity and specificity and could be applied to study the expression and locating of Sglt1 in the C.carpio.The Sglt1-P polyclonal antibody also could serve as an important research tool to study Sglt1 expression and transshipment activity of C.carpio.Meanwhile,the Sglt1-P polyclonal antibody could also be used to exploratively study the expression location and quantity of the Sglt1 transporters in other fishes.Keywords:
Polyclonal antibodies
Common carp (Cyprinus carpio) is one of the most important cultured fish in the world aquaculture industry. The aim of this study was to determine the phenotypic parameters of some body measurements in common carp (Cyprinus carpio L., 1758) reared in pools in Kirkuk province of Iraq. In this study, the means of body weight (BW), total length (TL), body depth (BD) and fork length (FL) in common carps were determined as, 2.097±0.034, 45.820±0.242, 14.446±0.240 and 38.040±0.210 for male and 2.050±0.033, 45.850±0.277, 14.120±0.154 and 37.960±0.227 for female (P>0.05), respectively. Besides, condition factor (K) was determined as 2.1829±0.0338 for male and 2.1383±0.0409 for females (P>0.05), respectively.
Common carp
Fish measurement
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Polyclonal anti-idiotypic antibodies to AIV were prepared from rabbits vaccinated with purified chicken anti-AIV antibodies, and isolated by aldehydated chicken red blood cells to bind unspecific chicken antibodies and to absorb rabbit-anti-chicken antibodies. The acquired antibodies were capable of agglutinating chicken red blood cells and inhibiting AIV in hemagglutinating weekly, and competed original AIV in binding chicken anti-AIV antibodies.
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2,6-dinitrotoluene (2,6-DNT) is the preferential toxicants stipulated by OECD and EPA of USA. The present study was conducted to determine the effect of 2,6-DNT chemical on acute toxicity and subacute toxicity in common carp (Cyprinus carpio). The 24h, 48h, 72h and 96h LC50 values of common carp by 2,6-DNT were 1.137±0.023mg/L, 0.830±0.024mg/L, 0.661±0.019mg/L and 0.479±0.020mg/L, respectively. The subacute experiment shows that C. carpio are rarely affected by 0.0251mg/L 2,6-DNT, but significantly affected by 0.0398, 0.0631, 0.0794mg/L 2,6-DNT in contrast to the controlled sample (p liver > kidney.
Common carp
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Common carp Cyprinus carpio, stressed by fish handling practices, responded with a decrease in cortisol secretion when temperature was lowered from 20 to 14° C within 3·5 h compared to those kept at 20° C.
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Common carp
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Juge-Aubry CE, Liang H, Lang J, Barlow JW, Burger AG. Synthesis and characterization of anti-idiotypic anti-thyroxine antibodies. Eur J Endocrinol 1994;130:107–12. ISSN 0804–4643 We injected rabbits with purified monoclonal murine immunoglobulin (IgG1) or polyclonal anti-thyroxine antibodies (anti-T 4 ) and polyclonal anti-triiodothyroacetic acid (anti-Triac) antibodies to stimulate the production of anti-idiotypic antibodies. Purified immunoglobulins from all five rabbits immunized with monoclonal primary antibodies were able to inhibit the interaction between [ 125 I]T 4 and the primary antibody. The preimmune sera were inactive. This effect was not due to endogenous T 4 contamination or contamination with the injected primary antibody. Half-maximal inhibition of binding of primary antibody with anti-idiotype was between 1.6 and 30 μg of total immunoglobulins. Addition of normal mouse IgG1 did not alter the inhibitory effect of the anti-idiotypic antibody. suggesting that this effect is specific. These anti-idiotypic antibodies reacted differently with different polyclonal antibodies, reflecting the heterogeneous nature of polyclonal antibody populations. Polyclonal antibodies were less effective in stimulating anti-idiotypic antibody production. One polyclonal anti-T 4 and one anti-Triac antibody produced weak anti-idiotypic antibody that had to be used at a concentration of > 600 μg of total immunoglobulins to be inhibitory. Both inhibited the binding of T 4 to the monoclonal anti-T 4 antibody. However, they were ineffective in inhibiting the function of their own antigen, the polyclonal anti-T 4 or anti-Triac antibody. We tested the most potent anti-idiotypic antibodies for their ability to compete with T 4 for other T 4 -binding proteins. Specific inhibition of T 4 binding to thyroid-binding globulin was observed with half-maximal effect at approximately 450 μg of total IgG. The antibody was negative when tested against Transthyretin, rat liver deiodinase type I, triiodothyronine cell uptake and liver cytoplasmic triiodothyronine binding. In conclusion, the technique described herein allows production of anti-idiotypic anti-T 4 , which can be useful in the characterization of the range of iodothyronine-binding sites involved in thyroid hormone action. AG Burger, Unité de la Thyroïde, Hôpital Cantonal Universitaire, 1211 Genève 4, Switzerland
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Purpose: In addition to conventional antibody Ig G1 in serum of camel,there are two subtypes of heavy chain antibody Ig G2 and Ig G3,they have the advantages of small molecular weight and strong stability,but the research and application of camel heavy chain antibody was certainly restricted since lacking of efficient detection techniques. It has some application prospect to prepare the polyclonal antibody that specific for camel heavy chain antibody. Method: In this experiment,camel plasma was obtained by Mararating lymphocytes from the camel whole blood with lymphocytes isolation kit,and then the three subtype antibody of camel was Mararated and purified through Protein G / A Resin column. After three times immunization of rabbit with camel Ig G2,the rabbit polyclonal antibodies were prepared in allusion to heavy chain antibody of camels,and its activity was identified. Result: The result of SDS- PAGE gel electrophoresis showed that camel subtype antibodies were Mararated and purified successfully. Indirect ELISA results showed the titer of polyclonal antibody was 1: 4 096. By combining Indirect ELISA and Western- blot determine the specificity of the antibody,we got that the polyclonal antibody has no any cross reaction with cattle Ig G and camel Ig G1,but has reaction with Ig G3. Conclusion: The experimental results show that we prepared camel heavy chain antibody specific polyclonal antibody successfully,and it will provide strong support for the development and utilization of camel antibody resources,as well as camel disease detection and research.
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Antibody titer
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The extant study was carried out to discern the effect of different acidic pH on the tissues of fishes. The experimental fish Cyprinus carpio were introduced into acidic waters (pH 5.0; 5.8; 6.6 and control 7.2) for 21 days for the histopathological exploration,
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The morphological variations between common carp Cyprinus carpio var. jian, Cyprinus carpio haermatopterus and the reciprocal F1 hybrids(Cyprinus carpio var. jian♀×Cyprinus carpio haermatopterus♂, and Cyprinus carpio var. jian♂×Cyprinus carpio haermatopterus♀) were studied by variance analysis and by three methods of multivariate analysis, based on their morphological characters. There were no significant differences among four groups based on the number of scales on the lateral line, scales above the lateral line, scales beneath the lateral line, spines of pectoral fins and ventral fin(P0.05) except for the number of spines of the dorsal fin, anal fin, and soft rays of the dorsal fin, anal fin, ventral fin and pectoral fins. The cluster results showed that the morphological traits were similar between the parent fish stocks, but quite different from those of the reciprocal F1 hybrids. The discriminant functions of the four stocks were established, and the discriminatory accuracies were 76.7%–96.7%(P1)and 85.2%–90.6%(P2).The total discriminatory accuracy was 87.5%. In the principal component analysis, nine principal components were constructed, and the cumulative contributory ratio was 68.94%. The study would be beneficial for identifying groups, and for determining genetic relationships and breeding.
Common carp
Dorsal fin
Fish fin
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Previously we quantitated plasma Abeta40 and Abeta42 levels using a combination of mouse monoclonal antibody 6E10 as a capture antibody and rabbit polyclonal antibodies specific to Abeta as detection antibodies. Recently, we have produced rabbit monoclonal antibodies (Rabmab) to Abeta40 and Abeta42. Currently there is a great interest in the measurement of plasma Abeta levels at intervals in AD and elderly non-demented controls. Reports showed conflicting data and the reason is not known. We hypothesize that differences in antibody epitopes and affinity may have contributed to the variable findings. We examined 40 AD or control plasma samples using 1) a combination of Rabmab to Abeta as capture antibodies and 4G8 mouse monoclonal antibody as detecting antibody; and 2) mouse monoclonal antibody 6E10 as a capture antibody and Rabmab to Abeta as detecting antibodies in sandwich ELISA. There was a significant relation between Abeta40 levels of Rabmab to Abeta40 and rabbit polyclonal antibody to Abeta40 using 4G8 as detecting antibody (r = .67; p < 0.001), and Rabmab to Abeta42 and rabbit polyclonal antibody to Abeta42 using 4G8 as detecting antibody (r = .97; p < 0.001). Similarly there was a relation between Abeta levels using mouse monoclonal antibody 6E10 as capture antibody and Rabmab or polyclonal antibody as detecting antibodies. When the relationship between the levels using 4G8 and 6E10 were compared, there was a significant relation in Abeta40 levels (r = .63; p < .001) but not in Abeta42 levels (r = .19; p < .24). The data show that differences in capture or detecting antibodies may contribute to different findings among published studies.
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