| Abstract: | 台灣土雞為國人喜好之禽肉，但台灣土雞繁殖性能不佳，其繁殖力與相關基因之關係仍不清楚。本研究的主要目的是選殖台灣土雞鐵蛋白重鏈（FHC）及麩胱甘?S轉移?A1（GST-A1）基因，並產製重組蛋白質及專一性抗體，研發檢驗的方法，以探討其與土雞產蛋性能的關係。高產蛋性能L2、低產蛋性能B品系土雞與紅羽土雞、來亨蛋雞，皆由中興大學所提供。前人利用雜合扣除法(Suppression subtractive hybridization, SSH)，由高繁殖力（L）及低繁殖力（B）台灣土雞之脂肪組織選殖出FHC及GST-A1二種表現差異之基因。含鐵蛋白（Ferritin）是由24個重鏈及輕鏈之次單位體所組成，為哺乳類之主要儲鐵蛋白質。麩胱甘?S轉移?（Glutathione S-transferase, GST）為抗氧化酵素。本試驗則利用反轉錄聚合?連鎖反應（reverse transcription-polymerase chain reaction；RT-PCR)的方法，分別從台灣土雞脂肪及肝臟組織RNA擴增此二特定全長cDNA，將其構築於原核細胞之表現質體後，並加以核?酸定序確認，於大腸桿菌中大量表現重組蛋白質(rFHC、rGST-A1)，再利用Ni-NTA大量純化重組蛋白質。RT-PCR分析的結果顯示，FHC廣泛的表現於腦、心、肺、胃、脾、胰、肝、腎、骨骼肌、脂肪、睪丸和卵巢等器官。GST-A1廣泛的表現於腦、肺、胃、肝、腎、脂肪、睪丸和卵巢等器官。以SDS-PAGE方法分析所得之重組蛋白質，結果顯示rFHC及rGST-A1分子量分別為26及27 kDa。利用紐西蘭白兔產製rFHC及rGST-A1多株抗體(polyclonal antibody)，以西方轉漬法測定其抗體專一性，並檢測台灣土雞脂肪組織FHC和GST-A1的含量，結果顯示FHC於高產土雞（L）之脂肪中表現量相較於低產品係（B）高，但GST-A1於此兩品係間則沒有差異。以酵素連結免疫分析法（Enzyme-linked immunosorbent assay, ELISA）偵測各品系雞隻血清之差異量。分析結果顯示，FHC於產蛋能力不同品系雞之血清間具有差異性，其中又以紅羽土雞血清含量最低，故FHC在血清中的含量和土雞的產蛋性能可能有關。
Taiwan Country Chicken is one of the favorite poultry in Taiwan. The reproductive ability of Taiwan Country Chicken is low and the relationship between reproduction and its related genes is not known. The main objective of this study was to clone ferritin heavy chain (FHC) and glutathione S-transferase A1 (GST-A1) genes from Taiwan Country Chickens and over-express the recombinant proteins to generate specific antibodies to investigate the relationship between these two genes and egg production in Taiwan Country Chickens. The Red-Feather Chickens, Leghorn Chickens and both high (L2) and low (B) egg production lines from Taiwan Country Chickens were provided by National Chung-Hsin University in Taichung. The differential expression of FHC and GST-A1 genes was previously identified in adipose tissues of high (L) and low (B) egg production lines of Taiwan Country Chickens using suppression subtractive hybridization (SSH) method. The ferritin, consisting of 24 heavy and light chains, is the major iron-storage protein. The glutathione S-transferase (GST) is an antioxidative enzyme. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the full-length cDNAs of FHC and GST genes from total RNAs of adipose and liver tissues in Taiwan Country Chickens. The cDNAs were cloned into prokaryotic expression vectors and sequence determined. The recombinant proteins, rFHC and rGST-A1, were over-expressed in E. coli system. These proteins were purified by Ni-NTA affinity chromatography. The results from RT-PCR indicated that FHC expressed ubiquitously in brain, heart, lung, stomach, spleen, pancreas, liver, kidney, skeletal muscle, adipose tissue, testis and ovary. GST-A1 expressed ubiquitously in brain, heart, lung, stomach, spleen, adipose tissue, testis and ovary. The results from SDS-PAGE analysis indicated that the estimated molecular weights for rFHC and rGST-A1 were 26 and 27 kDa, respectively. New Zealand rabbits were immunized to generate polyclonal antibodies against rFHC and rGST-A1. Western blot analysis was used to confirm the specificity of polyclonal antibodies and detect protein contents of FHC and GST-A1 in adipose tissues. The results indicated that the level of FHC protein was higher in L line than that in B line of Taiwan Country Chickens. However, there was no significant difference in GST-A1 protein level between these two lines. The sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine serum concentrations of FHC in various strains of chickens. The results indicated that serum FHC content was significantly lower in Red-Feather chicken compared to other strains. In conclusion, the FHC content in serum appears to be related to the egg production. |
