目的构建包含乙型肝炎病毒(hepatitis B virus,HBV)聚合酶TP区段的重组原核表达质粒,转化表达型大肠埃希菌,以自诱导培养方法获得可溶性表达,并对其进行鉴定。方法分析HBV(A型)聚合酶N-末端1-192 AA区域的DNA序列,通过网络工具(http://w...目的构建包含乙型肝炎病毒(hepatitis B virus,HBV)聚合酶TP区段的重组原核表达质粒,转化表达型大肠埃希菌,以自诱导培养方法获得可溶性表达,并对其进行鉴定。方法分析HBV(A型)聚合酶N-末端1-192 AA区域的DNA序列,通过网络工具(http://www.jcat.de/)在线优化,并在5′和3′端分别添加NdeⅠ和XhoⅠ限制性内切酶位点后,送英潍捷基(上海)贸易有限公司进行人工合成;将人工合成的TP-DNA双酶切后,插入pET32a(+)原核表达载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3)pLysS进行自诱导表达。结果重组原核表达质粒pET32a(+)/POL-TP-Opt经双酶切及测序证明构建正确;在表达菌的破菌上清中有特异性表达的蛋白,相对分子质量约20 000,经SDS-PAGE和Western blot鉴定,为可溶性的重组TP蛋白。结论通过自诱导培养方法,直接成功获得可溶性的重组TP蛋白,改变了长期以来依靠包涵体复性对TP蛋白相关功能进行研究的状况。展开更多
BACKGROUND:Paraquat(PQ) is an effective herbicide and is widely used in agricultural production,but PQ poisoning is frequently seen in humans with the lung as the target organ.Currently,there are many studies on lung ...BACKGROUND:Paraquat(PQ) is an effective herbicide and is widely used in agricultural production,but PQ poisoning is frequently seen in humans with the lung as the target organ.Currently,there are many studies on lung injury after PQ poisoning.But the kidney as the main excretory organ after PQ poisoning is rarely studied and the mechanisms of this poisoning is not very clear.In this study,we observed the expression of caspase-3 and livin protein in rat renal tissue after PQ poisoning as well as the therapeutic effects of ulinastatin.METHODS:Fifty-four Sprague-Dawley(SD) rats were randomly divided into three experimental groups:control group(group A),paraquat poisoning group(group B) and ulinastatin group(group C),with 18 rats in each group.Rats in group B and group C were administered intragastrically with 80mg/kg PQ,rats in group C were injected peritoneally with 100 000 U/kg ulinastatin once a day,while rats in group A were administered intragastrically with the same volume of saline as PQ.At 24,48,72 hours after poisoning,the expression of livin in renal tissue was detected by Westen blotting,the expression of caspase-3 was detected by immunohistochemistry,and the rate of renal cell apoptosis was tested by TUNEL detection.The histopathological changes were observed at the same time.RESULTS:Compared to group A,the expression of caspase-3 in the renal tissue of rats in groups B and C increased significantly at any time point.Compared with group B,the expression of caspase-3 in renal tissue of rats in group C decreased.Compared with group A,the expression of livin in renal tissue in rats of groups B and C increased significantly at any time point(P<0.01),especially in group C(P<0.01).TUNEL method showed that the rate of renal cell apoptosis index was higher in group B at corresponding time points than in group A(P<0.01),and was lower in group C at corresponding time points than in group B(P<0.01).CONCLUSION:UTI has a protective effect on the renal tissue of rats after paraquat poisoning through up-regulating the 展开更多
文摘BACKGROUND:Paraquat(PQ) is an effective herbicide and is widely used in agricultural production,but PQ poisoning is frequently seen in humans with the lung as the target organ.Currently,there are many studies on lung injury after PQ poisoning.But the kidney as the main excretory organ after PQ poisoning is rarely studied and the mechanisms of this poisoning is not very clear.In this study,we observed the expression of caspase-3 and livin protein in rat renal tissue after PQ poisoning as well as the therapeutic effects of ulinastatin.METHODS:Fifty-four Sprague-Dawley(SD) rats were randomly divided into three experimental groups:control group(group A),paraquat poisoning group(group B) and ulinastatin group(group C),with 18 rats in each group.Rats in group B and group C were administered intragastrically with 80mg/kg PQ,rats in group C were injected peritoneally with 100 000 U/kg ulinastatin once a day,while rats in group A were administered intragastrically with the same volume of saline as PQ.At 24,48,72 hours after poisoning,the expression of livin in renal tissue was detected by Westen blotting,the expression of caspase-3 was detected by immunohistochemistry,and the rate of renal cell apoptosis was tested by TUNEL detection.The histopathological changes were observed at the same time.RESULTS:Compared to group A,the expression of caspase-3 in the renal tissue of rats in groups B and C increased significantly at any time point.Compared with group B,the expression of caspase-3 in renal tissue of rats in group C decreased.Compared with group A,the expression of livin in renal tissue in rats of groups B and C increased significantly at any time point(P<0.01),especially in group C(P<0.01).TUNEL method showed that the rate of renal cell apoptosis index was higher in group B at corresponding time points than in group A(P<0.01),and was lower in group C at corresponding time points than in group B(P<0.01).CONCLUSION:UTI has a protective effect on the renal tissue of rats after paraquat poisoning through up-regulating the
