Two Biomarker Cytochrome P450 1A1 And P-glycoprotein In Mugilogobius Abei: CDNA Clone And Expression Induced By Norfloxacin

Antibiotic has been widely used in many applications,including medicine and agriculture.But it will cause some ecosystem and environment problems due to some abuse and unreasonable disposal.When antibiotic enter organisms,it would induce the body defensing and metabolizing,the response would be displayed at various levels such as molecules,cellular,organic and population,even ecosystem.The molecule and cellular response would be used as biomarker for Pollutants early detection.In this paper,we showed the full-length cDNA sequence of Mugilogobius abei(M.abei) CYP1A1 gene and partial sequence of P-gp gene.We proved that expression of CYP1A1 and P-glyeoprotein from liver tissues of M.abei can be up regulated by exposed to Rifampicin(RFP),Norfloxacin and water borne in field.The conclusion showed that M.abei would be useful indicator sentinel for contamination evaluation and monitoring in aquatic ecosystem.The full-length cDNAs of M.abei CYP1A1 gene were obtained using RT-PCR and RACE(rapid amplification of cDNA ends) PCR amplification of total RNA extracted from liver of a M.abei which were exposed by being injected withβ-NF.The result of sequence analysis indicated that the full length of M.abei CYP1A1 cDNA is 1943bp, containing an open reading frame(ORF) of 1552bp encoding a 516 amino acids protein whose deduced protein molecular weight is 58.71kDa.The cloned cDNA possesses all characteristic motifs of teleost CYP1A genes including the start codon,heme-binding region,arginine residue critical to enzymatic function and stop codon.Its deduced protein shares 90.4%,79.8%,68.3%,78.9%,73.2%,62.8%and 76.8%sequence identity with the putative proteins of Fundulus heteroclitus,Oryzias latipes,Danio rerio,Liza saliens,Oncorhynchus mykiss,Microgadus tomcod and Platichthys flesus CYP1A genes,respectively.A pair of degenerate primer is designed,thus a cDNA fragment of M.abei P-gp gene with a length of 741 bp is obtained,which has highly similarity with P-gp mRNA from other species.A 1665bp cDNA fragment is obtained by semi-nested PCR.using the primer designed according to the obtained sequence.A 741bp sequence of M.abei P-gp gene is cloned by modified degenerate PCR technology.We joint the two fragments using the Vetor VTI 10.2 suit and obtain a fragment of 1985bp,the deduced amino acid sequence of M.abei P-gp shows 90.1%identity with Fundulus heteroclitus.M.abei CYP1A1 and P-glycoprotein expression was divided by beta-actin expression to determine relative expression.RT-PCR analysis showed that CYP1A1 mRNA was at a significantly higher level in the liver of M.abei injected intraperitoneally with 5mg/kg body weightβ-Naphthoflavone(βNF),5mg/kg body weight norfloxacin after 72h,respectively.These results suggested that the CYP1A1 and P-gp gene was up regulated by the two chemical.Expression of CYP1A1 and P-glycoprotein from liver tissues of M.abei can be up regulated by exposed to 50,20,10 or 5ug/L norfloxacin.The expression of both CYP1A1 and P-gp can be detected after 24,72 and 168h exposed in field.The fish cytochrome P450 1A1 and P-glycoprotein gene are useful biomarkers in assessing Pharmaceuticals and Personal Care Products (PPCPs) such as norfloxacin antibiotics.In order to allow M.abei for assessment of PPCPs of environment using quantitative protein methods,we should explore to express M.abei's CYP1A1 and P-gp gene recombinant protein and produced its specific antibodies.The further research will facilitate to M.abei as a model for PPCPs toxic evaluation and monitoring in aquatic ecosystem.