恶性疟原虫乳酸脱氢酶的表达、纯化、单克隆抗体的制备及鉴定
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摘要
疟疾是一种严重危害人类健康的虫媒性传染病，尤其在热带、亚热带地区流行广泛。据WHO统计，全球每年有近3-5亿人感染疟疾，其中约300万人死亡。在我国也有24个省、市及自治区不同程度地存在疟疾流行。据我国卫生部疟疾委员会最新资料显示，南部地区如云南、广西、海南等省疟疾发病率较往年都有不同程度的提高，因此，疟疾的防治始终是寄生虫病研究的重点之一。疟疾的诊断是疟疾防治工作中的一个重要环节，目前疟疾诊断技术正朝着简便、快速、灵敏、特异及结果易于判定的方向发展，而以单抗为基础的Dipstick免疫层析技术具有以上优点，应用前景广泛。本研究旨在利用疟原虫的乳酸脱氢酶(lactate dehydrogenase, LDH)为诊断靶抗原，通过对该蛋白进行克隆、表达，制备单抗，并筛选、配对建立反应模式等系列研究，开发用于疟疾快速诊断的免疫层析检测试剂盒。
研究中，首先将恶性疟原虫的LDH(Plasmodium falciparum lactate dehydrogenase, LDHpf)基因克隆至pET-23a(+)表达载体中，构建pET23-LDH重组质粒。重组质粒在BL21中得到高效表达，SDS-PAGE显示表达蛋白分子量约为33KDa，与先前报道的疟原虫LDH分子量理论值(31-36Kda)基本符合。表达产物冰浴超声破菌，发现目的蛋白大部分溶于上清中，为可溶性表达，密度扫描显示目的蛋白占菌体总蛋白的41.2％。采用Q-Sepharose High Performance阴离子交换树脂柱层析对pET23-LDH重组蛋白进行纯化，SDS-PAGE电泳经凝胶光密度图象分析系统检测主蛋白纯度可达89.1％，浓度为400μg／ml。用纯化的重组蛋白免疫BALB／c小鼠制备免疫血清，ELISA结果显示免疫小鼠血清与pET23-LDH
表达产物出现明显反应，而对空载体PET一23a表达产物无反应。western
blot分析表明特异性区带出现在33kDa处，而空载体诱导后表达的产物
无区带出现。进一步用该免疫血清与恶性疟原虫和间日疟原虫反应，
western b10t结果表明免疫血清能识别恶性疟原虫和间日疟原虫33KDa
处虫源蛋白，而与未感染的红细胞不发生交叉反应，提示pET23一LDH重
组蛋白具有良好的抗原性和免疫原性。
以纯化后的LDH重组蛋白免疫BALB/c小鼠，取其脾细胞与小鼠骨髓
瘤细胞SPZ/0进行融合，融合细胞用队T培养基作选择性培养。采用间
接ELISA法筛选阳性杂交瘤细胞株，获得n株能分泌高效价和高特异性
的抗LDH重组蛋白单抗的杂交瘤细胞株。工g类与亚类鉴定结果为2Bll、
3C9、3DIO、6D3、7D2、IE7、6G7、IC6、2D7重链类型为1 9 Gl亚类，
轻链为K型;2F12重链类型为1 9 GZ亚类，轻链为K型;2C6重链类
型为1 gGI亚类，轻链为人型。n株单抗培养上清的ELISA效价为1:
100~1:400，腹水效价为l:6400一1:51200。从而可为恶性疟原虫免
疫胶体金快速诊断试剂盒的研制提供抗体。
利用饱和硫酸按纯化后的n株单抗，建立了同时诊断恶性疟原虫和
间日疟原虫的G工以法。两次筛选的结果显示以3C9包被抗体，IE7标记
抗体为GICA较适反应模式。以此反应模式对重组抗原Ing/m1，sng/ml，
10ng/ml，100 ng/ml，500 ng/ml进行测定，其最低检测量为5 ng/ml，
并且结果呈现较好的梯度，说明反应线颜色的深浅与抗原含量成正比。
用制备的免疫层析条对30例恶性疟病人血样、40例间日疟病人血样及
100例正常人血样进行检测，恶性疟的敏感性为83.3%，间日疟敏感性为
57.5%，特异性均为98%。
虽然在敏感性较国外试剂盒还有差别，但提示我们LDHPf为检测靶
抗原是可行的，如在单抗的制备、GIGC工艺上做进一步完善，开发出同
时诊断恶性疟原虫和间日疟原虫的试剂盒指日可待。
Malaria is a severe and harmful parasitic disease which broadly spreads in tropical and sub-tropical regions of the world 0 The statistical results by WHO indicate that about 300-500 million people suffer from malaria each year among which 3 million patients die of this disease, Malaria also occurs in most parts of China, of which the incidence of this disease in Southern China such as Yunnan, Guangxi and Hainan has been increasing0 Hence, Diagnosis and treatment of malaria has become one of the most critical roles in parasitic disease researcho The current diagnostic technique of malaria is proceeding to the aspect which is simple, rapid, sensitive specific and easy to be determined The Dipstick immunoaffinity chromatography, a technique which is based on the monoclonal antibody, possesses the advantages above and is a promising and effective way for the diagnosis of malaria. The rationale of this study is to utilize the plasmodium LDH( pLDH) as the targeting antigen to
prepare the monoclonal antibodies, and to develop a diagnosis kit, which recognizes both falciparum malaria and tertian malaria using the exclusive method. The method is fast, affordable and effective, which meets the requirement for the malaria diagnosis that the tertian malaria is the dominant type in China .
In our present study, we clone LDHpf cDNA into PET-23a(+) expression vector, obtaining PET23-LDH recombination construct which is highly expressed in BL21 bacteria. The molecular weight of this protein is about 33kDa, which is consistent with the reported 31-36kDa of this protein in plasmodium . The expression product is dissolved in the supernatant after
-4-
the bacteria is lysed by ultrasonic, and is 41.2 percent of the total bacteria proteins o Upon the purification of the combination protein by Q-Sepharose High Performance negative ion-exchange chromatography, the purity is about 89.1 percent measured by SDS-PAGE and gel image analysis system ?And the concentration is 400ng/ml0 We further immunize mouse using the recombination protein to prepare the antiserumo ELASA result shows a very strong reaction between the antiserum and the pET23-LHD expression product hi contrast, there is no reaction between the antiserum and the expression product from the pET-23 empty vector control o Western blot analysis indicates that there exists a 33kDa specific band in pET23-LDH containing bacteria lysis, however, there is no positive band in pET-23a vector containing bacteria 0 Polyclonal antibodies thus prepared could specifically recognize a 33kDa protein from P.falciparum and P.vivax as revealed by Western blot, without reacting to normol hunman red blood cell, which sug
gests that the pET-LDH recombination protein possesses a high antigenicityo
limmunize mouse using the recombination protein , We adopt its spleen cell mouse marrow SP2/0, Amalgamation cell use HAT culture medium carry through cultive The identification results for Ig type and sub-type indicate that 2BIK 3C9 3DIO 6D3 7D2 1E7 6G7C6 and 2D7 are IgGl for heavy chain and K for light chain; 2F12 is IgG2 for heavy chain and K for light chain; and 2C6 is IgGl for heavy chain and A for light chain o The ELASA titers of the culture cell supernatant from all the 11 clones range from 1:100 to 1:400 and those of ascites are from 1:6400 to 1:51200.
GICA method which recognizes both falciparum malaria and tertian malaria is then established using the monoclonal antibodies purified by saturated ammonium sulfateo The results from two rounds of screen show an optimal reaction pattern, in which the 3C9 represents the coating antibody while the 1E7 is the marker antibody ?This reaction pattern is applied to detect different concentration of recombination antigen: Ing/mK 5ng/mK
-5-
l0ng/mK l00ng/ml and 500ng/ml, in which the lowest concentration that could be detected is 5ng/ml, and the results show a fine gradient which suggests a direct proportional relationship between the reaction color and the antigen amount 0 Thirty cases of falciparum
研究中，首先将恶性疟原虫的LDH(Plasmodium falciparum lactate dehydrogenase, LDHpf)基因克隆至pET-23a(+)表达载体中，构建pET23-LDH重组质粒。重组质粒在BL21中得到高效表达，SDS-PAGE显示表达蛋白分子量约为33KDa，与先前报道的疟原虫LDH分子量理论值(31-36Kda)基本符合。表达产物冰浴超声破菌，发现目的蛋白大部分溶于上清中，为可溶性表达，密度扫描显示目的蛋白占菌体总蛋白的41.2％。采用Q-Sepharose High Performance阴离子交换树脂柱层析对pET23-LDH重组蛋白进行纯化，SDS-PAGE电泳经凝胶光密度图象分析系统检测主蛋白纯度可达89.1％，浓度为400μg／ml。用纯化的重组蛋白免疫BALB／c小鼠制备免疫血清，ELISA结果显示免疫小鼠血清与pET23-LDH
表达产物出现明显反应，而对空载体PET一23a表达产物无反应。western
blot分析表明特异性区带出现在33kDa处，而空载体诱导后表达的产物
无区带出现。进一步用该免疫血清与恶性疟原虫和间日疟原虫反应，
western b10t结果表明免疫血清能识别恶性疟原虫和间日疟原虫33KDa
处虫源蛋白，而与未感染的红细胞不发生交叉反应，提示pET23一LDH重
组蛋白具有良好的抗原性和免疫原性。
以纯化后的LDH重组蛋白免疫BALB/c小鼠，取其脾细胞与小鼠骨髓
瘤细胞SPZ/0进行融合，融合细胞用队T培养基作选择性培养。采用间
接ELISA法筛选阳性杂交瘤细胞株，获得n株能分泌高效价和高特异性
的抗LDH重组蛋白单抗的杂交瘤细胞株。工g类与亚类鉴定结果为2Bll、
3C9、3DIO、6D3、7D2、IE7、6G7、IC6、2D7重链类型为1 9 Gl亚类，
轻链为K型;2F12重链类型为1 9 GZ亚类，轻链为K型;2C6重链类
型为1 gGI亚类，轻链为人型。n株单抗培养上清的ELISA效价为1:
100~1:400，腹水效价为l:6400一1:51200。从而可为恶性疟原虫免
疫胶体金快速诊断试剂盒的研制提供抗体。
利用饱和硫酸按纯化后的n株单抗，建立了同时诊断恶性疟原虫和
间日疟原虫的G工以法。两次筛选的结果显示以3C9包被抗体，IE7标记
抗体为GICA较适反应模式。以此反应模式对重组抗原Ing/m1，sng/ml，
10ng/ml，100 ng/ml，500 ng/ml进行测定，其最低检测量为5 ng/ml，
并且结果呈现较好的梯度，说明反应线颜色的深浅与抗原含量成正比。
用制备的免疫层析条对30例恶性疟病人血样、40例间日疟病人血样及
100例正常人血样进行检测，恶性疟的敏感性为83.3%，间日疟敏感性为
57.5%，特异性均为98%。
虽然在敏感性较国外试剂盒还有差别，但提示我们LDHPf为检测靶
抗原是可行的，如在单抗的制备、GIGC工艺上做进一步完善，开发出同
时诊断恶性疟原虫和间日疟原虫的试剂盒指日可待。
Malaria is a severe and harmful parasitic disease which broadly spreads in tropical and sub-tropical regions of the world 0 The statistical results by WHO indicate that about 300-500 million people suffer from malaria each year among which 3 million patients die of this disease, Malaria also occurs in most parts of China, of which the incidence of this disease in Southern China such as Yunnan, Guangxi and Hainan has been increasing0 Hence, Diagnosis and treatment of malaria has become one of the most critical roles in parasitic disease researcho The current diagnostic technique of malaria is proceeding to the aspect which is simple, rapid, sensitive specific and easy to be determined The Dipstick immunoaffinity chromatography, a technique which is based on the monoclonal antibody, possesses the advantages above and is a promising and effective way for the diagnosis of malaria. The rationale of this study is to utilize the plasmodium LDH( pLDH) as the targeting antigen to
prepare the monoclonal antibodies, and to develop a diagnosis kit, which recognizes both falciparum malaria and tertian malaria using the exclusive method. The method is fast, affordable and effective, which meets the requirement for the malaria diagnosis that the tertian malaria is the dominant type in China .
In our present study, we clone LDHpf cDNA into PET-23a(+) expression vector, obtaining PET23-LDH recombination construct which is highly expressed in BL21 bacteria. The molecular weight of this protein is about 33kDa, which is consistent with the reported 31-36kDa of this protein in plasmodium . The expression product is dissolved in the supernatant after
-4-
the bacteria is lysed by ultrasonic, and is 41.2 percent of the total bacteria proteins o Upon the purification of the combination protein by Q-Sepharose High Performance negative ion-exchange chromatography, the purity is about 89.1 percent measured by SDS-PAGE and gel image analysis system ?And the concentration is 400ng/ml0 We further immunize mouse using the recombination protein to prepare the antiserumo ELASA result shows a very strong reaction between the antiserum and the pET23-LHD expression product hi contrast, there is no reaction between the antiserum and the expression product from the pET-23 empty vector control o Western blot analysis indicates that there exists a 33kDa specific band in pET23-LDH containing bacteria lysis, however, there is no positive band in pET-23a vector containing bacteria 0 Polyclonal antibodies thus prepared could specifically recognize a 33kDa protein from P.falciparum and P.vivax as revealed by Western blot, without reacting to normol hunman red blood cell, which sug
gests that the pET-LDH recombination protein possesses a high antigenicityo
limmunize mouse using the recombination protein , We adopt its spleen cell mouse marrow SP2/0, Amalgamation cell use HAT culture medium carry through cultive The identification results for Ig type and sub-type indicate that 2BIK 3C9 3DIO 6D3 7D2 1E7 6G7C6 and 2D7 are IgGl for heavy chain and K for light chain; 2F12 is IgG2 for heavy chain and K for light chain; and 2C6 is IgGl for heavy chain and A for light chain o The ELASA titers of the culture cell supernatant from all the 11 clones range from 1:100 to 1:400 and those of ascites are from 1:6400 to 1:51200.
GICA method which recognizes both falciparum malaria and tertian malaria is then established using the monoclonal antibodies purified by saturated ammonium sulfateo The results from two rounds of screen show an optimal reaction pattern, in which the 3C9 represents the coating antibody while the 1E7 is the marker antibody ?This reaction pattern is applied to detect different concentration of recombination antigen: Ing/mK 5ng/mK
-5-
l0ng/mK l00ng/ml and 500ng/ml, in which the lowest concentration that could be detected is 5ng/ml, and the results show a fine gradient which suggests a direct proportional relationship between the reaction color and the antigen amount 0 Thirty cases of falciparum
引文
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