| Growth hormone (GH), also known as somatotropin, is a single strand polypeptide hormone synthesized and secreted by eosinocytes in the anterior pituitary gland, with prolactin (PRL) and galactagogin (RL) belong to the same gene family, widely existing in various kinds of vertebrates. It is essential for growth stimulation and body metabolism. Recombinant bovine growth hormone (rbGH) is widely used in livestock production of the United States and other countries nowadays, since it can increase milk yield in bovines. With Decisions, European Union and Canada ban to use it, based on its negative effects on bovines, including serious mastitis, limp and dysgenesis. Furthermore, there are higher risks of cancer within human consumption of milk and dairy products which are coming from rbGH treated animals. However, illegal utility of the hormone is inevitable for gaining huge profits.The need to develop analytical methods detects rbGH treatments, in order to counteract or prevent rbGH misuse. Unfortunately, the high similarity of the recombinant forms with the natural hormone makes it extremely difficult to develop analytical methods to directly reveal the presence of rbGH. Therefore, indirect analytical approaches, which involve the exploitation of biomarkers, could represent a possible alternative. This research mainly contains two parts:1) preparation for monoclonal antibodies against rbGH, establishment of double sandwich ELISA methods to detect serum GH, serum insulin-like growth factor (IGF-1) and milk IGF-1;2) establishing methods to detect peripheral blood leukocyte differentially expressed genes targets.Since there are still no data of studies on rbGH treatment in buffalos, we chose buffalo as model animal, treated8lactating buffalos with biweekly injections of Korean LG-Life science rbGH, totally five times for ten weeks (every2weeks for a cycle, totally5cycles). Collection of the samples was performed on days2,5,9,14of each cycle of rbGH administration for blood; on days2,7,14of the first and last cycle for milk; on days2and14of each cycle for leukocytes.In order to distinguish highly similar forms of rbGH and natural bGH, this research was prepared monoclonal antibody2A3and3G6with high specificity and sensitivity, and2A3 could totally distinguish rbGH from natural bGH. Three double sandwich ELISA methods also have been established to detect serum GH, serum IGF-1and milk IGF-1. These methods were verified by statistical methods of sensitivity, specificity, accuracy and repeatability, which confirmed the effectiveness of these methods. Moreover, their limit of detection (LOD) could reach0.089ng/ml,20ng/ml,0.025ng/ml, respectively.Results of those double sandwich ELISA tests showed:1) compared with the control groups, an extraordinary increase of GH serum levels on Day2of each cycle in rbGH treated animals (p<0.001), followed by a rapid decrease in the subsequent days, even though significant differences with the controls were observed until Day5(p<0.001), the value returned to the basal levels on Day9and14. These results promoted serum GH levels as a good and reliable indicator of rbGH treatment, but the rapid decrease after the peak limits the exploitable period for sample collection.2) IGF-1concentrations promptly rose in serum on Day2of each cycle in rbGH treated animals (p<0.001), and on Day5or9reached to peak, despite the value of serum IGF-1has declined, on an average, they were still significantly higher in the treated animals on Day14(p<0.01). IGF-1concentration in serum would allow to rely on a longer period for inspection, within two weeks after each rbGH injection. Furthermore, an analysis plan involving a number of animals belonging to the same farm would make the discovery of high levels of serum IGF-1a strong indicator for rbGH treatment.3) IGF-1levels in milk were found to be significantly higher during the whole cycle, with peaks (more than2.3times compared to the controls) on days2(p<0.001) and7(p<0.01). High IGF-1concentrations in milk of treated buffalos, rather than to be considered an rbGH inspection indicator, should be regarded as a potential concern in term of food safety, due to potential harm to public health with regard of high levels of IGF1in treated bovine milk.Differentially expressed genes have been obtained in the peripheral blood leukocytes of rbGH treated buffalos and controls by microarray analysis. Among the genes,110genes showed up-regulation or down-regulation. Thus,10genes have been screened as being the most relevant in terms of difference in expression levels between treated animals and controls, including ABCG1, DQA1, DQA5, DQB, DLC, Ferritin-like heavy chain, Integrin β-fragment, KKCC, TKDP3and SUCLG. Subsequently, expression levels of those ten genes from microarray analysis, of ten genes belonging to the somatotropic axis (GHR, IGF-1, IGF-2, IGFBP-1,-2,-3,-4,-5,-6, IGF-1R), of five GHR5’-UTR variants (1B,1C1,1C3,1D,1I), have been detected in the peripheral blood leukocytes of rbGH treated buffalos and controls by means of Real-time PCR. These results revealed that exogenous GH significantly influenced the expression of parts of the investigated genes (IGFBP-1, p<0.001; DQA5, p<0.01; DQB p<0.05; DLC p<0.001; Ferritin-like heavy chain, p<0.05; Integrin β-fragment, p<0.05). Among them, only DQA5expression level seemed to be strongly triggered and sustained by the hormonal treatment, whereas it was hard to detect in non-treated buffalos (but only one mastitis buffalo in the control group has been detected high expression level of DQA5). In a word, DQA5also can be used as biological target for identification of rbGH injection, but this gene expression analysis should be used with caution for the emergence of the false positive.In this research, we identified whether the animal model of buffalo injected rbGH through establishing those detection methods of GH, IGF-1and gene expression analysis. Moreover, the potential biomarkers (including serum GH, serum IGF-1, milk IGF-1and25genes of buffalo peripheral blood leukocytes) have been inspected, confirming the feasibility of those established methods. This study will lay a foundation of promoting the standardization methods for the effective regulation rbGH usage, provide technical supports for in-depth development of rbGH related research, and bring positive influence to the monitoring of our country’s food safety in the future. |
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