Detection Of Ciguatoxin, Cloning Of Collagen Binding Protein Cna Gene Of Staphylococcus Aureus And Its Expression

In the first part,we developed a mouse toxicity assay and a cytotoxicity bioassay to detect ciguatoxin and confirmed these methods were more effective than Cigua-Check^?.Male KunMing strain mice were applied to confirm the dosage of ciguatoxin (P-CTX-1).One mouse unit is about 9.10 ng P-CTX-1,95%of confidence interval is between 8.67 ng and 9.56 ng;A curve relation between the doses of ciguatoxin and death time of male Kunming strain mouse was established and the curve equation was Y=244.9X^-1.3937,where x means the toxin dosage and Y means lethal time.The method was validated at 0.1 ng/g,0.2 ng/g and 0.3 ng/g.The overall recovery varied from 35.23%to 58.06%.The method is simple and accurate,and can be applied to detect the actual toxicity in samples.Neuroblastoma cells in culture were used to detect ciguatoxin.Ciguatoxin could be detected with a sensitivity of 0.004μg/kg in 10 h;while the commercial kit showed a notable positive result at the level above 2μg/kg.Later the method was validated at the ciguatoxin levels of 0.05μg/kg,0.10μg/kg and 0.15μg/kg in samples.Validation results were as the follows:the overall recovery varied from 42.03%to 60.00%,and the relative standard deviation(RSD) is between 1.65%and 5.37%.Sensitivity and accuracy of the assay were considered to meet the requirements of ciguatoxin detection.In the second part,in order to investigate the effect of collagen binding proteins as vaccine against cow mastitis caused by Staphylococcus aureus,The results of research was described as follows:1.Staphylococcus aureus isolated from milk with mastitis and S.aureus isolate with typical biochemical reactions,highly toxic was used as the material for genome extraction.2.Using PCR,we amplified the cna gene(1509 bp) which encodes collagen binding proteins(Cna),and then cloned into the expression vector pET-28a to obtain pET-28a-cna.The sequence of cna gene was 98%similarity at both the nucleotide acid and amino acid levels to those of the reference strain in GenBank database (accession No:M81736).3.The plasmid pET-28a-cna was transformed into E.coli BL21(DE3)competent cells.The bacteria were induced by IPTG(1 mmol/L) and analyzed by SDS—PAGE. Approximately 55 KDa protein was expressed.4.Collagen binding proteins was purified by Ni-NTA His Bind Purification Kit and the concentration was detected with BCA Kit to be is 1 mg/mL.