Construction Of Recombinant Human Androgen Receptor Gene Yeast Cells And Application In The Detection Of Androgen Residue

As a kind of steroid hormone,androgen can promote protein synthesis and accelerate muscle growth.Androgens are used as feed additives in order to accelerate the effect of animal fattening.However,androgens in food animals may enter the human body through the food chain and other forms,causing harm such as early puberty in children,cancer and endocrine disorders.Therefore,the detection of androgen residues has attracted more and more attention.The existing residue analysis methods such as chromatographic analysis and immunoassay cannot solve the low-dose and multi-component addition of androgens,and the detection cost is high and the operation is complicated.Androgen receptor reporter gene detection is a multi-residue detection method,which responds to the ’cocktail effect’ caused by the low-dose and multi-component addition of various androgens.It is low-cost and easy to operate,and suitable for food safety supervision and inspection.In this study,the plasmid pRR-hAR-5ARE/Lac Z constructed with human androgen receptor,the five-linked androgen response elements(5ARE)and the reporter gene(Lac Z)downstream of the promoter sequence was transferred into yeast cell W303-1A.Nine androgens including DHT,testosterone,methyltestosterone,nandrolone,androstenedione,trenbolone,methandrostenolone,boldenone and stanozolol stimulated recombinant yeast cells to verify their pharmacological activity and optimize the time of stimulation.Then,according to the error prone PCR,random mutanted receptor which increased androgens activity compared with AR-WT were screened.The conditions and time limit for the preservation of recombinant yeast cells were explored by freeze-drying technique.Finally,samples such as feed,soil and water were collected from farms in Jiangsu,and then the recombinant yeast cells were used for preliminary screening after pretreatment.The positive samples were detected by HPLC-MS for identification and quantitation,in order to explore the feasibility of recombinant yeast cells in the application of androgens residue detection.The results are as follows:1.Under the action of 9 androgens,with the increase of androgens concentration,the level of β-galactosidase in recombinant androgen receptor yeast cells also increased,showing a good dose-effect relationship.EC50 range is between 32.51 and 624.76 μg/L.2.The β-galactosidase level of recombinant yeast cells stimulated by DHT was detected at different times.Starting from 6 h,the β-galactosidase level began to increase.Starting at 12 h,it tends to level off and reach saturation.3.Double-points mutant G581R/D831E with increased androgens activity was screened.G581R and D831E were constructed by site-directed mutagenesis in order to better analyze the changes of mutants in androgens reactivity in yeast cells.Different concentrations of androgens were used to stimulate AR-WT and mutants recombinant yeast cells to detect the level of β-galactosidase.The results showed that the basal β-galactosidase level of G581R/D831E and G581R increased,the maximum response value decreased,and so the signal window decreased.With the signal window is unchanged,the activity of D831E on androgens is higher than WT.The ECso range is between 5.06 and 69.01 μg/L.4.Yeast cells were preserved by freeze-drying technology,and their pharmacological activity was verified in a while.The results showed that the freeze-dried powder of recombinant yeast cell could be stored at-20℃ for at least 40 days with the same pharmacological activity.5.The 63 samples collected from the farm were initially screened with recombinant yeast cells,and a total of 12 positive samples were detected.Four androgens were detected in positive samples by HPLC-MS.The results of HPLC-MS were predicted to DHTEq and the correlation was compared with that of recombinant yeast.The results showed that they had good correlation within the detection range,which verified the accuracy and feasibility of recombinant yeast cells in the application of androgens residue detection.This study provides a theoretical basis for the establishment of a convenient and accurate method for the detection of androgen drug residues in the environment based on the construction of recombinant human androgen receptor gene yeast cells.