Saxitoxin(STX)was conjugated to bovine serum albumin(BSA) as immune antigen.Inoculated mouse with STX-BSA,gained one hybridoma cell strains secreting monoclonal antibodies against STX stably.The subclass of the McAb was IgM.The titer of the McAb was 1∶102400 in ascites according to the indirect ELISA determination.The affinity constant was 3.71×106 L/mol.
The complete antigens were prepared by the mixed anhydride coupling method in which Neurotoxic Shellfish Poisoning Toxin PbTx-2 was conjugated with BSA and OVA respectively.A hybridoma cell line steadily secreting monoclonal antibodies against PbTx-2,coded 4B5,was obtained by fusing immunized SP2/0 cells with spleen cells from 8 weeks old BALB/c mice immunized with PbTx-BSA conjugate and subcloning for 3 to 4 cycles.The titer of the McAb in the ascites analyzed by indirect ELISA was 1:5.12×104,the antibody subclass was IgM,affinity constant was 2.32×107mol/L.
Objective To establish the method for quality control of Shenjiao Shengxue granule. Methods Ren Shen, Huang Qi and He Shou Wu were identified by TLC. The content of astragaloside iv was determined by HPLC-ELSD. Results The spots of TLC were clear and concentrative. There was a good linear relationship for astragaloside iv with the ranges of 0.021-0.063 μg(r=0.9998). Their average recoveries were 98.95% (RSD=1.27). Conclusion This method is sensitive, accerate, reproducible and exclusive.
Prion diseases are neurodegenerative diseases,harmful to animal and human health.Mechanisms of prion diseases are not fully understood.Systems of prion replications in vitro are used to studying prion pathogenesis,however,mock environment in vitro for studying conversion mechanisms from normal cellular prion proteins to disease-associated scrapie prion proteins is relatively difficult and very important.Cell-free conversion assay,cell-lysate conversion assay,Protein Misfolding Cyclic Amplification,Autocatalytic conversion assay,several methods of prion conversion in vitro were described,and discussed them in reflecting prion propagation in vivo,a lot of target samples for studying Prion disease were provided,in order to facilitate to further study prion pathogenesis in the future.
Silkworm chrysalis intoxication may be related to microbial contamination,deteriorate,protein denaturation and produced toxins.The various kinds of contaminated microbial and produced toxins are responsible for this intoxicatio.In this study,bacterium of silkworm chrysalis from the market of Changchun was analyzed in order to ensure silkworm chrysalis edible safety.
Five gene fragments from E.coli O157∶H7,Staphylococcus aureus and C.botulinum were connected by SOE PCR via linker sequence encoding five amino acids(G-P-G-P-G).After digested with restriction nuclease NcoI and XhoI,the linked gene was cloned into pMD18-T vector.The resultant recombinant plasmid,termed F5,was transformed into E.coli DH5α and then linked into expression vector pET-22b(+),termed F5-22b,was transformed into host strain E.coli BL21(DE3).The recombinant toxin expressed was induced by IPTG and identified by SDS-PAGE.The expressed product was immuned rabbits to obtain serum antibody.In this way fusion gene F5(Hc-VT1B-SEA-VT2B-SEB)and recombinant plasmid F5-22b were constructed.The sequenced results of the fusion gene F5 was consistent with the predicted gene sequences.The sequence encoding the mature fusion protein of the F5 toxin gene was 2 937bp,encoding 979 amino acids.The molecular weight of recombinant fusion toxin protein was 112.369 ku.The expressed protein amounted to 9.90% of the total protein of E.coli BL21(DE3)after induced by IPTG(1mmol/L)for 4 hours.Two expressed products including soluble protein in pET-22b and inclusion body were obtained.Serum antibodies against five toxins were obtained from rabbits immunized with expressed products.ELISA and agar diffusion assay showed that the antibodies had high reactive specificity and sensitivity with five toxins.The findings of this experiment would lay foundations for the establishment of the broad-spectrum immunologic methods to detect food poisoning borne bacteria or toxins.
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