【摘要】：為探究安氏隱孢子蟲(Cryptosporidium andersoni)ATP結(jié)合盒轉(zhuǎn)運蛋白基因(CaABC2)對營養(yǎng)物質(zhì)的轉(zhuǎn)運功能,以安氏隱孢子蟲基因組作為模板,設(shè)計合成CaABC2基因特異性引物,進行PCR擴增,獲得CaABC2基因。將克隆的重組質(zhì)粒CaABC2基因與真核表達質(zhì)粒pEGFP-C1雙酶切后再連接,構(gòu)建重組真核質(zhì)粒載體pEGFP-C1-CaABC2;再采用脂質(zhì)體轉(zhuǎn)染法將重組質(zhì)粒pEGFP-C1-CaABC2轉(zhuǎn)入小鼠腸上皮細胞(Intestinal epithelial cells,IEC),檢測不同組IEC細胞對總膽固醇、還原型谷胱甘肽、葡萄糖和堿性磷酸酶的轉(zhuǎn)運效果。結(jié)果表明:擴增出778bp的CaABC2基因,測序分析與預期片段大小一致;重組真核質(zhì)粒載體pEGFP-C1-CaABC2轉(zhuǎn)染小鼠IEC細胞后,觀察到轉(zhuǎn)染后的對照組和轉(zhuǎn)染組IEC有綠色熒光;檢測到在細胞內(nèi)液中,轉(zhuǎn)染組、對照組和空白組的總膽固醇濃度分別為15.99±0.12、11.80±0.35和12.43±0.16 mmol/L;還原型谷胱甘肽濃度分別為15.24±0.44、10.48±0.35和11.04±0.75mmol/L;在細胞外液中,轉(zhuǎn)染組、對照組和空白組的總膽固醇濃度分別10.67±0.17、14.82±0.06和15.84±0.17 mmol/L;還原型谷胱甘肽濃度分別為10.20±0.79、14.60±0.45和15.60±2.50mmol/L。轉(zhuǎn)染組總膽固醇、還原型谷胱甘肽濃度均顯著大于空白組和對照組(P0.05),而在細胞外液中,轉(zhuǎn)染組總膽固醇、還原型谷胱甘肽的濃度均顯著小于空白組和對照組(P0.05)。在細胞內(nèi)液和外液中,空白組與對照組總膽固醇、還原型谷胱甘肽的濃度之間均未見顯著性差異(P0.05);轉(zhuǎn)染組、對照組和空白組之間的葡萄糖和堿性磷酸酶濃度均沒有統(tǒng)計學差異(P0.05)。CaABC2基因具有協(xié)助轉(zhuǎn)運總膽固醇、還原型谷胱甘肽、葡萄糖和堿性磷酸酶的作用,其中轉(zhuǎn)運總膽固醇效果最為明顯。
[Abstract]:In order to investigate the transport function of (Cryptosporidium andersoni) ATP binding cassette transporter gene (CaABC2) from Cryptosporidium andersoni, the specific primers of CaABC2 gene were designed and synthesized using Cryptosporidium Angiosporidium genome as template, and the CaABC2 gene was obtained by PCR amplification. The cloned recombinant plasmid CaABC2 gene was digested with eukaryotic expression plasmid pEGFP-C1 and then ligated. The recombinant eukaryotic plasmid vector pEGFP-C1-CaABC2; was constructed and the recombinant plasmid pEGFP-C1-CaABC2 was transfected into mouse intestinal epithelial cells (Intestinal epithelial cells,IEC) by liposome transfection. The transport effects of different IEC cells on total cholesterol, reduced glutathione, glucose and alkaline phosphatase were detected. The results showed that the CaABC2 gene of 778bp was amplified and sequenced, and the size of the CaABC2 gene was the same as expected. After the recombinant eukaryotic plasmid pEGFP-C1-CaABC2 was transfected into mouse IEC cells, the green fluorescence of IEC was observed in the control group and the transfected group, and the green fluorescence was detected in the intracellular fluid. The total cholesterol concentrations in transfection group, control group and blank group were 15.99 鹵0.121.80 鹵0.35 and 12.43 鹵0.16 mmol/L;, respectively, and the concentrations of reduced glutathione were 15.24 鹵0.4410.48 鹵0.35 and 11.04 鹵0.75 mmol / L, respectively. The total cholesterol concentrations in the control group and the blank group were 10.67 鹵0.17 鹵14.82 鹵0.06 and 15.84 鹵0.17 mmol/L;, respectively, and the glutathione concentrations were 10.20 鹵0.79 鹵14.60 鹵0.45 and 15.60 鹵2.50 mmol / L, respectively. The concentrations of total cholesterol and reduced glutathione in transfection group were significantly higher than those in blank group and control group (P0.05), while in extracellular fluid, the concentrations of total cholesterol and reduced glutathione in transfection group were significantly lower than those in blank group and control group (P0.05). There was no significant difference in the concentration of total cholesterol and glutathione between the blank group and the control group (P0.05). There was no significant difference in glucose and alkaline phosphatase concentrations between the control group and the blank group (P0.05). The CaABC2 gene could facilitate the transport of total cholesterol, reduced glutathione, glucose and alkaline phosphatase. Among them, the effect of total cholesterol transport is the most obvious.
【作者單位】： 安徽農(nóng)業(yè)大學動物科技學院;安徽農(nóng)業(yè)大學茶與食品學院;
【基金】：安徽省自然科學基金項目(1708085MC81) 國家自然科學基金項目(31001019)
【分類號】：S852.723

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