The Study On The Construction Of Recombinant Yeast Cells Regulated By Tetracycline And The Screening Of Tetracycline Residues In Pig Urine

Objective:The recombinant gene yeast cells regulated by tetracycline response element(TRE)were constructed to rapidly detect the residues of tetracycline antibiotics in animal food.Methods:1.The GPD promoter was amplified from yeast DNA extract by PCR and inserted into yeast expression vector of pYESTrp2,to construct yeast expression vector driven by GPD promoter,pGPD.2.The double-stranded DNA of V5 antigen epitope was synthesized and inserted into the pGPD successfully.The tetracycline repressor protein(Tet R)gene was amplified by PCR,using plasmid of pcDNA6/TR as template,and fused with V5 antigen epitope DNA of pGPD.Then GPD-driving TR yeast expression vector was transformed into the yeast cell W303-1A.Finally,the expression of TetR gene was identified by western blot method.3.Based on the pMP206 of vector,the tetracycline response element(TRE)was inserted between the CYC 1 promoter and the reporter gene Lac Z(?-galactosidase gene),to construct Lac Z yeast reporter vector,pyTeton-Lac Z,regulated by TRE,and this vector was transformed into the TR high-expressing yeast cell.4.The promoter CYC1(cytochrome oxidase gene)of pyTeton-Lac Z plasmid was replaced by strong promoter GAL1 which amplified by PCR using pYESTrp2 as template.The Lac Z gene in the new yeast reporter vector was driven by GAL1 and regulated by TRE.The vector was transformed into the TR high-expressing yeast cell.After that,the positive clones were induced by tetracycline,and the sensitivity and the specificity were analyzed.5.The pig urine samples of 146 were from a Yangzhou pig farm,the tetracycline antibiotics in urine were detected by the recombinant yeast cell method.Finally,the comparative analysis with gold standard method of ELISA method was carried out.Results:1.The results of PCR indicated that the genes of GPD,and tetR gene had been transformed into yeast cells successfully.The size of the fragment was in accordance with the expectation.The analysis of western blot showed that the tetR gene had successfully expressed in the yeast cells and the size of the protein was 22 KD.2.It was found by the results of PCR that the GAL1-driving and TRE regulating Lac Z yeast reporter vector had been successfully constructed.The constructed recombinant yeast cells showed significant dose-response relationship with tetracycline antibiotics,and without any dose effect relationship with non-tetracycline antibiotics,indicating that the recombinant yeast cells have good sensitivity and specificity.3.The comparison between ELISA and the recombinant yeast methods,found that there was significant difference between positive rates of the two methods for detecting tetracycline antibiotics(x2=22.56,P=1.881×10-6<0.01).By using ROC curve analysis,the sensitivity was 76.36%.However,its specificity and coincidence rates are not too high,43.96%and 56.16%,and Youden index was 0.203,positive likelihood ratio(+LR)was 1.363,negative likelihood ratio(-LR)was 0.538,the positive predictive value was 45.16%,the negative predictive value was 75.47%.The combined effect of mixed tetracyclic antibiotics can be found by recombinant yeast method and ELISA method.However,recombinant yeast is more sensitive to the combined action of tetracycline antibiotics mixture than ELISA.Conclusion:The construction of recombinant yeast cells(W303-1A/TR-GAL1-Teton-Lac Z)is more sensitive to the combined effect of tetracycline antibiotics mixtures,and a rapid screening of tetracycline antibiotics residues in animal urine,however,the specificity and sensitivity need further research to be improved.