本文關(guān)鍵詞：柴胡皂苷D人工抗原的合成及單克隆抗體的制備　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 柴胡皂苷D(SSD) 單克隆抗體 酶聯(lián)免疫吸附分析法(ELISA)

【摘要】：研究目的:柴胡是《傷寒雜病論》中應(yīng)用比較廣泛和重要的藥物之一,本實(shí)驗(yàn)的主要目的是合成柴胡皂苷D(SSD)的人工抗原與人工包被原,制備中藥活性成分SSD的單克隆抗體,利用SSD單克隆抗體建立相應(yīng)的酶聯(lián)免疫吸附分析方法,檢測含柴胡的中藥復(fù)方中SSD含量,為中藥復(fù)方質(zhì)量控制提供新的技術(shù)方法,也為后續(xù)對(duì)經(jīng)方中柴胡的作用機(jī)制和機(jī)理的研究奠定基礎(chǔ)。研究方法:(1)采用高碘酸鈉氧化法合成SSD的人工免疫抗原(SSD-BSA)和人工包被原(SSD-OVA),并且使用薄層色譜法對(duì)合成產(chǎn)物進(jìn)行鑒定;(2)用合成的SSD-BSA多次免疫BALB/c小鼠后,通過ELISA方法測定免疫小鼠抗SSD血清的效價(jià)和特異性;(3)取出免疫小鼠的脾細(xì)胞與小鼠骨髓瘤細(xì)胞SP2/0,采用PEG法進(jìn)行融合,再進(jìn)行單克隆細(xì)胞株的篩選,采用腹水誘導(dǎo)法生產(chǎn)大量的單克隆抗體;(4)利用制備的SSD單克隆抗體建立ELISA方法,測試該方法的特異性、靈敏度、交叉反應(yīng)率、回收率、準(zhǔn)確性等相關(guān)指標(biāo);(5)利用建立SSD免疫分析方法,檢測五種含有柴胡的復(fù)方中SSD含量。研究結(jié)果:(1)薄層色譜法鑒定的結(jié)果說明,合成的產(chǎn)物SSD-BSA和SSD-OVA偶聯(lián)成功;小鼠的血清效價(jià)在1:10000以上,標(biāo)準(zhǔn)競爭抑制曲線表明,血清中的抗體可與SSD產(chǎn)生特異性結(jié)合。取免疫的小鼠脾細(xì)胞進(jìn)行細(xì)胞融合,篩選出能分泌抗SSD抗體的單克隆細(xì)胞株�；谥苽涞腟SD單克隆抗體,建立相應(yīng)的ELISA分析法。(2)方法學(xué)考察結(jié)果顯示,在156.25～5000ng/mL范圍內(nèi),包被原SSD-OVA以1:4000的濃度稀釋,腹水以1:100000的濃度稀釋,得到SSD單克隆抗體的抑制率與線性關(guān)系曲線,回歸方程為:y=-0.158In(x)+1.7693,R2=0.9856。IC50為 425.81 ng/mL。回收率在98.15～104.37%(均值為101.19%),板內(nèi)差異8.16%,板間差異13.78%。通過ELISA法測試SSD與其它中藥活性成分反應(yīng)的交叉反應(yīng)率。結(jié)果顯示SSD與其他成分的交叉反應(yīng)率均小于0.10%。說明制備的SSD單克隆抗體有很好的特異性。研究結(jié)論及意義:本實(shí)驗(yàn)首次成功的合成了 SSD的人工抗原,為SSD單克隆抗體的篩選和制備以及相應(yīng)免疫方法的建立奠定基礎(chǔ);首次篩選出SSD的單克隆株;首次制備出SSD的單克隆抗體;首次利用生產(chǎn)出的SSD的單克隆抗體建立酶聯(lián)免疫吸附分析方法。這些研究可以為中藥復(fù)方中柴胡的質(zhì)量監(jiān)控提供新的檢測方法,而且為藥物有效成分的分離研究、中藥復(fù)方有效物質(zhì)基礎(chǔ)的研究、體內(nèi)代謝、作用機(jī)理等的研究奠定基礎(chǔ)。
[Abstract]:Objective: Bupleurum chinense is one of the most widely used and important drugs in Typhoid complex Disease. The main purpose of this experiment is to synthesize the artificial antigen and artificial envelope of saikosaponin (DX SSDs). The monoclonal antibody of Chinese medicine active ingredient SSD was prepared and the corresponding enzyme-linked immunosorbent assay (Elisa) method was established by using SSD monoclonal antibody to detect the content of SSD in traditional Chinese medicine compound containing Bupleurum chinensis. To provide a new technical method for the quality control of traditional Chinese medicine compound. It also lays a foundation for further study on the mechanism and mechanism of Bupleurum chinensis in Jingfang. Methods: the artificial immune antigen (SSD-BSA) of SSD was synthesized by sodium periodate oxidation method. And artificial bag coated with SSD-OVA). The synthetic products were identified by TLC. (2) after immunizing BALB/c mice with synthetic SSD-BSA for several times, the titer and specificity of anti-#en3# serum were determined by ELISA method. The spleen cells of immunized mice and mouse myeloma cells SP2 / 0 were taken out and fused by PEG method, then the monoclonal cell lines were screened, and a large number of monoclonal antibodies were produced by ascites induction. (4) the ELISA method was established by using the SSD monoclonal antibody. The specificity, sensitivity, cross reaction rate, recovery rate and accuracy of the method were tested. SSD immunoassay was established to determine the content of SSD in five compounds containing Bupleurum chinense. The synthesized product SSD-BSA and SSD-OVA were successfully coupled. The titer of mouse serum was more than 1: 10000. The standard competitive inhibition curve showed that the antibody in the serum could bind to SSD specifically. The spleen cells of immunized mice were fused to each other. The monoclonal cell lines which can secrete anti SSD antibody were screened. Based on the SSD monoclonal antibody, the corresponding ELISA assay was established. In the range of 156.25 ~ 5000ng / mL, the original SSD-OVA was diluted with 1: 4000 and ascites with 1: 100000. The linear curve of inhibition rate of SSD monoclonal antibody was obtained. The regression equation was 1. 7693. The IC50 of R2N 0.9856. IC50 was 425.81 ng / mL. The recovery rate was 98.15 ~ 104.37 (mean value was 101.19) and the intraplate difference was 8.16%. The cross-reaction rate between SSD and other active components of traditional Chinese medicine was measured by ELISA method. The results showed that the cross-reaction rate between SSD and other components was less than 0.10. The monoclonal antibody to SSD prepared by MMT has good specificity. Conclusion and significance of the study:. In this experiment, the artificial antigen of SSD was successfully synthesized for the first time. It will lay a foundation for the screening and preparation of monoclonal antibodies against SSD and the establishment of corresponding immune methods. The monoclonal strain of SSD was screened for the first time. The monoclonal antibody of SSD was prepared for the first time. Enzyme linked immunosorbent assay (Elisa) was established by using monoclonal antibody of SSD for the first time. These studies can provide a new method for quality control of Bupleurum chinensis. It also lays a foundation for the study of the separation of active components of drugs, the basis of effective substances of traditional Chinese medicine compound, the metabolism in vivo, the mechanism of action, and so on.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張華平;;芪歸四逆散聯(lián)合恩替卡韋治療乙型肝炎37例[J];光明中醫(yī);2017年05期

2 李響;張豐川;;小柴胡湯加減治療尋常痤瘡肝郁脾虛證30例[J];河南中醫(yī);2017年03期

3 曲騫;吳煜;;經(jīng)方治療結(jié)直腸癌并發(fā)癥驗(yàn)案3則[J];江蘇中醫(yī)藥;2017年03期

4 王怡;沈健;魏夫榮;;四逆散加減聯(lián)合雷貝拉唑腸溶膠囊治療胃食管返流性咳嗽患者的效果及對(duì)IL-8、SP、MCT水平的影響[J];中國生化藥物雜志;2017年02期

5 王彩鳳;;大柴胡湯聯(lián)合消炎利膽片治療肝膽濕熱型慢性膽囊炎的療效觀察[J];現(xiàn)代醫(yī)藥衛(wèi)生;2017年04期

6 邵元欣;吳春麗;王興臣;;基于“少陽為樞”理論運(yùn)用小柴胡湯治療頭痛驗(yàn)案舉隅[J];湖南中醫(yī)雜志;2017年02期

7 張潔;李建波;楊秀麗;馬鳴;李巧敏;張莉;;小柴胡湯加減治療癌性發(fā)熱的臨床研究[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2017年06期

8 張輝;吳昊;田紀(jì)鳳;鄭瑾;范玉貞;楊建剛;任秦有;;大柴胡湯“辨病”治療消化道腫瘤癌性發(fā)熱的經(jīng)驗(yàn)體會(huì)[J];中國中醫(yī)急癥;2017年02期

9 廖韓鵬;;柴胡桂枝干姜湯合理中丸加針灸治療膽汁反流性胃炎的臨床研究報(bào)道[J];內(nèi)蒙古中醫(yī)藥;2017年02期

10 王耀榮;魯曉玲;;小柴胡湯合蒿芩清膽湯治療化療所致惡心嘔吐的臨床療效[J];實(shí)用臨床醫(yī)藥雜志;2017年02期

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