伏马菌素B_1和赭曲霉毒素A噬菌体单链抗体库的构建及重组抗体蛋白的原核表达
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摘要
真菌毒素(Mycotoxin)是各种真菌产生的一系列有毒次级代谢产物,广泛存在于自然界中,并污染粮食谷物及各种农产品。真菌毒素对人类健康和畜牧业发展已经构成潜在威胁,它不仅降低产品质量,而且可带来严重的食源性中毒问题,引起人或动物的急性、慢性中毒。动物试验表明,摄入被真菌毒素污染的饲料后,就会引起人或动物免疫抑制、组织坏死、肝肾损伤、繁殖障碍、致癌致畸等病理变化,影响人和动物的身体健康。自然界存在的真菌毒素种类繁多、毒性复杂,由于其污染广泛、毒害范围广,在全球食品安全中受到高度关注。
为了防止污染真菌毒素的食品及农畜产品直接或间接的进入人类食物链,加强对真菌毒素的检测是十分必要的。近年来,真菌毒素的检测技术快速发展,主要是以高效液相色谱法(HPLC)为主的色谱学检测技术及以酶联免疫吸附法(ELISA)为主的免疫学检测技术。色谱法检测灵敏度高,但样品质量要求较高、前处理操作复杂；而以抗原抗体为基础的免疫学方法,由于其操作简便、快速的优点一直被人们所青睐。现今,专家学者专注于优化和改良ELISA方法,以提高ELISA方法的检测性能。
基因工程抗体技术是在充分认识抗体的基因结构和功能的基础上,应用DNA重组技术和蛋白质工程技术发展起来的第三代抗体。基因工程抗体的研究意义在于可以有目的的对抗体基因进行切割、拼接、修饰等一系列操作,获得具有特异功能的改造抗体。与多克隆抗体和单克隆抗体相比,基因工程抗体制备具有时间周期短、操作简便、规模生产成本低廉等优点。单链抗体(ScAb)是基因工程抗体中的一种,它的基因不易丢失,容易保存,作为传统单克隆抗体的改良品,ScAb的具有较好的应用前景,已成为免疫检测技术发展的一个方向。本研究以两种常见的真菌毒素,赭曲霉毒素A(OTA)和伏马菌素Bl(FB1)为对象,应用分子生物学技术及噬菌体展示技术,制备两种真菌毒素的单链抗体,为其免疫学应用与发展奠定基础。
试验I伏马菌素B1和赭曲霉毒素A完全抗原的合成与鉴定
为了获得FB1和OTA的抗体基因,本试验采用戊二醛一步法将FBl与牛血清白蛋白(BSA)和卵清蛋白(OVA)两种载体蛋白偶联,制备FBl完全抗原FB1-BSA和FB1-OVA;同时用碳二亚胺法合成OTA完全抗原OTA-BSA和OTA-OVA。随后,分别采用紫外吸收光谱法(UV)、凝胶电泳法、傅里叶红外光谱法(IR)、基质辅助激光分析电离飞行时间质谱法(MALDI-TOF-MS)四种方法鉴定人工抗原偶联效果并测定偶联比。将FB1-BSA和OTA-BSA分别作为免疫抗原免疫BALB/c小鼠,采用间接酶联免疫吸附法(i-ELISA), FB1-OVA和OTA-OVA分别作为固相包被抗原,测定小鼠抗血清滴度。结果表明,OTA和FBl完全抗原合成成功。MALDI-TOF-MS测定完全抗原FB1-BSA、FB1-OVA. OTA-BSA和OTA-OVA偶联比分别为10：1、5：1、3：1和9：1。i-ELISA测定经FB1-BSA和OTA-BSA免疫的小鼠血清效价均可达到1：1.024×105。试验制备的完全抗原以及经免疫的小鼠可用于下一步试验。
试验Ⅱ伏马菌素B1单链抗体可变区基因克隆与噬茵体抗体蛋白表达
提取FB1杂交瘤细胞F3细胞总RNA,反转录合成cDNA第一条链,应用PCR技术分别将FB1鼠源抗体重链可变区基因(VH)和轻链可变区基因(VL)克隆出来,并经过重叠延伸PCR (SOE-PCR),通过柔性多肽Linker接头(Gly4Ser)3,按VH-Linker-VL方式拼接成FBl单链抗体可变区基因(ScFv)片段。随后将ScFv基因重组入pCANTAB5E载体中,转化至大肠杆菌(E. coli)宿主菌TGl中,应用噬菌体展示技术,以FB1-OVA为固相包被抗原进行免疫亲和筛选,应用Phage-ELISA和ScFv-ELISA鉴定和筛选阳性噬菌体重组单链抗体。将阳性pCANTAB5E-FB1ScFv质粒转化入表达菌株E. coli HB2151中,经IPTG诱导,获得FB1特异性噬菌体抗体(ScAb).结果显示,设计鼠源单克隆抗体的重链、轻链可变区基因引物,成功扩增获得FB1鼠源抗体的重链、轻链基因片段,大小为300～400bp。SOE-PCR将VH和VL由Linker拼接成FB1ScFv,大小为700～800bp。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,经过辅助噬菌体M13K07感染,构建得到的FBl噬菌体重组单链抗体库,抗体滴度约为2.1×1015cfu·mL-1。经过五轮免疫亲和富集和筛选,成功筛选得到5株可分泌FBl特异性噬菌体单链抗体的菌株。核苷酸序列鉴定显示该序列为717bp,编码239个氨基酸。在M13K07辅助噬菌体的辅助下,pCANTAB5E噬菌体质粒可产生含有FBl单链噬菌体基因组的噬菌体颗粒,并以融合的形式表达在噬菌体表面。在E.coli HB2151中,FB1ScFv基因在翻译过程中形成独立的抗体蛋白,以胞周间质形式形成可溶性蛋白。
试验Ⅲ赭曲霉毒素A单链抗体可变区基因克隆与噬茵体抗体蛋白表达
用OTA-BSA免疫BALB/c小鼠,免疫效价达到要求后,提取小鼠脾总RNA,反转录合成cDNA第一条链,应用PCR技术分别克隆OTA鼠源抗体VH和VL,并经过SOE-PCR,通过柔性多肽Linker接头,按VH-Linker-VL方式拼接成OTA ScFv片段。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,应用噬菌体展示技术,以OTA-OVA为固相包被抗原进行免疫亲和筛选,应用Phage-ELISA和ScFv-ELISA鉴定和筛选阳性噬菌体重组单链抗体。将阳性pCANTAB5E-OTA ScFv质粒转化入表达菌株E. coli HB2151中,经IPTG诱导,获得OTA特异性ScAb。结果显示,设计鼠源单克隆抗体的重链、轻链可变区基因引物,成功扩增获得OTA鼠源抗体的VH和VL,大小为300～400bp。SOE-PCR将VH和VL由Linker拼接成ScFv,大小为700～800bp。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,经过辅助噬菌体M13K07感染,构建得到的OTA噬菌体重组单链抗体库,抗体滴度约为2.65×1016cfu·mL-1。经过三轮免疫亲和富集和筛选,成功筛选得到3株可分泌OTA特异性噬菌体单链抗体的菌株。核苷酸序列鉴定显示该序列为738bp,共编码246个氨基酸。在pCANTAB5E噬菌体质粒中,OTA ScFv以胞周间质形式形成可溶性蛋白。
试验Ⅳ伏马菌素B1和赭曲霉毒素A单链抗体特性鉴定
应用蛋白免疫印迹(WB)和i-ELISA方法分别鉴定OTA与FB1两种真菌毒素ScAb的免疫活性。应用i-ELISA分别测定OTA与FB1ScAb的效价。应用间接竞争酶联免疫吸附试验(ci-ELISA)建立竞争抑制标准曲线。分别测定OTA和FB1ScAb的灵敏度,并测定OTA, FB1ScAb与参试毒素FB2、FB3、AFB1、ZEA和DON交叉反应率,以反映其特异性。结果显示,WB和i-ELISA均显示OTA与FB1ScAb具有良好的免疫活性。FB1-OVA抗原最佳工作浓度为5μg·mL-1, ScAb最佳稀释度为1:1600; OTA-OVA抗原最佳工作浓度为10μg-mL-1, ScAb最佳稀释度为1：800；FB1ScAb抗体滴度达到104; OTA ScAb抗体滴度达到103; ci-ELISA法绘制FB1竞争抑制曲线,FB1浓度为0.31～20μg-mL-1范围内曲线线性关系良好,线性方程为y=-16.663x+117.98(R2=0.9674), FB1ScAb对FB150%抑制浓度4.08μg·mL-1;同样方法绘制OTA竞争抑制曲线,OTA浓度为0.31～10μgmL-1范围内线性关系良好,线性方程为y=-16.075x+116.84(R2=0.9797),OTA ScAb对OTA的50%抑制浓度4.16μg·mL-1。FB1ScAb对FB1同种属的FB2、FB3的交叉反应抑制率分别为71.7%和87.6%,而与DON、AFB1、OTA和ZEA交叉反应抑制率较小,说明该FB1ScAb对FB1具有较好的特异性；OTA ScAb对FBi、DON、AFB1和ZEA交叉反应抑制率较小,说明该OTA ScAb对OTA具有较好的特异性。
Mycotoxins are a range of toxic secondary metabolites produced by various funguses and the contamination of grains and all kinds of agricultural products, which exist in nature widely. Mycotoxins have been a potential threat to human health and the development of animal husbandry that may not only reduce the quality of the products, but also lead to serious foodborne poisoning and cause people or animals to acute or chronic poisoning. Animal tests showeded that the animals that took in mycotoxins contaminated feed would have some pathological changes, such as immune suppression, tissue necrosis, liver and kidney damage, reproductive disorder, carcinogenesis and cacogenics and so on, and make direct or indirect impact on the health of humans and animals. Many kinds of mycotoxin exist in nature with complex toxicities. They have caused highly concerns over global food security owing to the widely pollution and toxicity. In order to prevent mycotoxins contaminated food and animal products from the human food chain, directly or indirectly, strengthening the detection of mycotoxins is essential. In recent years, the detection technologies for mycotoxins have been developed rapidly, such as the chromatographic detection technology mainly based on high-performance liquid chromatography (HPLC) and immunology detection technology based on enzyme-linked immunosorbent assay (ELISA). Though high sensitivity, chromatography analytical method has shortcomings, such as the high quality requirement for sample and complex pretreatment operation, which make it difficult to be applied in actual production extensively. Compared to this, immunological method, based on the antigen-antibody has been favored by people due to its easy operation and fast advantages. Now, some experts and scholars focus on the various aspects of optimization and improvement of ELISA method in order to improve the detection performance.
Genetically engineered antibody is the third generation of antibody that developed on the application of recombinant DNA technology and protein engineering technology and based on gene structure and function of the antibody. The significance of the genetically engineered antibody is to obtain the specific engineered protein with a series of operations, such as cutting, splicing and modification for antibody genes. Compared with polyclonal antibody and monoclonal antibody, genetically engineered antibody have its advantages, for example, short time prepare, simple operation, scale production and low cost. The Single-chain antibody (ScAb) is a member of genetic engineering antibody, which has been regarded as one direction of immunoassay technology development. As the improved product of traditional monoclonal antibody, ScAb has a better application prospect because of its stable and conserving easily. In this study, two kinds of ScAb against two common mycotoxins, ochratoxin A (OTA) and fumonisin B1(FB1) were obtained with molecular biology technique and phage display technique, so as to achieve a breakthrough both on theory and on technology, and would be the basic work for its immunoassay application and development.
Test I Synthesis and characterization of complete antigen for fumonisin B1and ochratoxin A
In order to obtain FB1and OTA antibody genes, FB1was coupled with carrier proteins ovalbumin (OVA) and bovine serum albumin (BSA) respectively, by one step glutaraldehyde method, while two complete antigens FB1-OVA and FB1-BSA were synthesized. OTA was coupled with OVA and BSA respectively to synthesized complete antigens OTA-BSA and OTA-OVA by carbodiimide method. Ultraviolet absorption spectroscopy (UV), gel electrophoresis, fourier transforms infrared spectroscopy (IR) and matrix-assisted laser absorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to determine the coupled effects and coupling ratios of the complete antigens, respectively. FB1-BSA and OTA-BSA were used to immunize BALB/c mice, and FB1-OVA and OTA-OVA were used as solid phase coating antigens, respectively. The indirect enzyme-linked immunosorbent assay (i-ELISA) was used to determine the titer of anti-serum from mice. The results showed that carrier proteins coupled with FB1and OTA successful, respectively. The coupling ratios of FB1-BSA, FB1-OVA, OTA-BSA and OTA-OVA were10:1,5:1,3:1and9:1by MALDI-TOF-MS method, respectively. The titers of anti-serum form the mice immunized FB1-BSA or OTA-BSA could be reached1:1.024×105determined by i-ELISA method, respectively. The complete antigens and the mice immunized could be used for the next test.
Test II The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against fumonisin B1
A total RNA was extracted from hybridoma cell line F3that secreted FB1monoclonal antibody. The first strand of cDNA was amplified by reverse transcription PCR (RT-PCR). The heavy chain and light chain variable region genes(VH and VL) were amplified by PCR used cDNA as the template, respectively. The FB1single chain antibody variable region gene fragment (ScFv) was splicing by overlap extension PCR (SOE-PCR) using a flexible the polypeptide (Gly4Ser)3as DNA linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into Escherichia coli (E. coli) host strain TG1and then rescued by M13K07helper phage. Biopanning and screening, FB1-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of FB1ScAb with IPTG. The results showed that FB1murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were348and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was717bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TG1, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.1×1015cfu·mL-1. After five times biopanning enrichment and screening, five strains that could secrete the FB1-specific phage single-chain antibody were selected successfully. Transformed cells were then infected with M13K07helper phage to rescue the phagemid with its ScFv gene insert. Recombinant phages which were produced contain a single-stranded DNA copy of the phagemid and antibody ScFv gene and display one or more copies of the recombinant antibody at their tips. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.
Test IH The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against ochratoxin A
A total RNA was extracted from spleen cell of mice immunized by OTA-BSA. The first strand of cDNA was amplified by RT-PCR. The VH and VL were amplified by PCR used cDNA as the template, respectively. The OTA ScFv was splicing by SOE-PCR using a linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into E. coli TGI and then rescued by M13K07helper phage. Biopanning and screening, using OTA-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of OTA ScAb with IPTG. The results showed that OTA murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were369and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was738bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TGI, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.65×1016cfu·mL-1. After five times biopanning enrichment and screening, three strains that could secrete the OTA-specific phage single-chain antibody were selected successfully. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.
Test IV Characterization of single-chain antibody against fumonisin B1and ochratoxin A
The immune activities of OTA and FB1recombinant ScAb were detected by Western blotting (WB) and i-ELISA, respectively. The titers of OTA and FB1recombinant ScAb were detected by i-ELISA, respectively. The sensitivities and competitive inhibition standard curves of OTA and FB1recombinant ScAb were established by competitive indirect enzyme-linked immunosorbent assay (ci-ELISA), respectively. And the specificities and cross-relativities of OTA and FB1ScAb to test toxins were determined using FB2, FB3, AFB1, ZEA and DON, respectively. The results showed that good immune activities of OTA or FB1ScAb to antigen were proofed through WB and i-ELISA method. The best working concentration of FB1-OVA antigen was5μg·mL-1and the best dilution of FB1ScAb was1:1600. The FB1ScAb titers reached104. The FB1competitive inhibition curve was established by ci-ELISA method, the linear equation y=-16.663x+117.98(R2=0.9674) and50%inhibition concentration (IC50) for OTA was4.08μg·mL-1, ranging from0.31～20μg·mL-1. The cross-reactivity inhibition rates of FB1ScAb to FB2and FB3were71.7%and87.6%, and the significant cross-reactivity with DON, AFB1, OTA and ZEA. The FB1ScAb has the better activity, sensitivity and specificity. The best working concentration of OTA-OVA antigen was10μg·mL-1and the best dilution of OTA ScAb was1:800. The OTA ScAb titers reached103. The OTA competitive inhibition curve was established by ci-ELISA method, the linear equation y=16.075x+116.84(R2=0.9797) and IC5o for OTA was4.16μg·mL-1, ranging from0.31～10μg·mL-1. The OTA ScAb had little cross-reactivity with DON, AFB1and ZEA. The OTA ScAb has better activity, sensitivity and specificity.
为了防止污染真菌毒素的食品及农畜产品直接或间接的进入人类食物链,加强对真菌毒素的检测是十分必要的。近年来,真菌毒素的检测技术快速发展,主要是以高效液相色谱法(HPLC)为主的色谱学检测技术及以酶联免疫吸附法(ELISA)为主的免疫学检测技术。色谱法检测灵敏度高,但样品质量要求较高、前处理操作复杂；而以抗原抗体为基础的免疫学方法,由于其操作简便、快速的优点一直被人们所青睐。现今,专家学者专注于优化和改良ELISA方法,以提高ELISA方法的检测性能。
基因工程抗体技术是在充分认识抗体的基因结构和功能的基础上,应用DNA重组技术和蛋白质工程技术发展起来的第三代抗体。基因工程抗体的研究意义在于可以有目的的对抗体基因进行切割、拼接、修饰等一系列操作,获得具有特异功能的改造抗体。与多克隆抗体和单克隆抗体相比,基因工程抗体制备具有时间周期短、操作简便、规模生产成本低廉等优点。单链抗体(ScAb)是基因工程抗体中的一种,它的基因不易丢失,容易保存,作为传统单克隆抗体的改良品,ScAb的具有较好的应用前景,已成为免疫检测技术发展的一个方向。本研究以两种常见的真菌毒素,赭曲霉毒素A(OTA)和伏马菌素Bl(FB1)为对象,应用分子生物学技术及噬菌体展示技术,制备两种真菌毒素的单链抗体,为其免疫学应用与发展奠定基础。
试验I伏马菌素B1和赭曲霉毒素A完全抗原的合成与鉴定
为了获得FB1和OTA的抗体基因,本试验采用戊二醛一步法将FBl与牛血清白蛋白(BSA)和卵清蛋白(OVA)两种载体蛋白偶联,制备FBl完全抗原FB1-BSA和FB1-OVA;同时用碳二亚胺法合成OTA完全抗原OTA-BSA和OTA-OVA。随后,分别采用紫外吸收光谱法(UV)、凝胶电泳法、傅里叶红外光谱法(IR)、基质辅助激光分析电离飞行时间质谱法(MALDI-TOF-MS)四种方法鉴定人工抗原偶联效果并测定偶联比。将FB1-BSA和OTA-BSA分别作为免疫抗原免疫BALB/c小鼠,采用间接酶联免疫吸附法(i-ELISA), FB1-OVA和OTA-OVA分别作为固相包被抗原,测定小鼠抗血清滴度。结果表明,OTA和FBl完全抗原合成成功。MALDI-TOF-MS测定完全抗原FB1-BSA、FB1-OVA. OTA-BSA和OTA-OVA偶联比分别为10：1、5：1、3：1和9：1。i-ELISA测定经FB1-BSA和OTA-BSA免疫的小鼠血清效价均可达到1：1.024×105。试验制备的完全抗原以及经免疫的小鼠可用于下一步试验。
试验Ⅱ伏马菌素B1单链抗体可变区基因克隆与噬茵体抗体蛋白表达
提取FB1杂交瘤细胞F3细胞总RNA,反转录合成cDNA第一条链,应用PCR技术分别将FB1鼠源抗体重链可变区基因(VH)和轻链可变区基因(VL)克隆出来,并经过重叠延伸PCR (SOE-PCR),通过柔性多肽Linker接头(Gly4Ser)3,按VH-Linker-VL方式拼接成FBl单链抗体可变区基因(ScFv)片段。随后将ScFv基因重组入pCANTAB5E载体中,转化至大肠杆菌(E. coli)宿主菌TGl中,应用噬菌体展示技术,以FB1-OVA为固相包被抗原进行免疫亲和筛选,应用Phage-ELISA和ScFv-ELISA鉴定和筛选阳性噬菌体重组单链抗体。将阳性pCANTAB5E-FB1ScFv质粒转化入表达菌株E. coli HB2151中,经IPTG诱导,获得FB1特异性噬菌体抗体(ScAb).结果显示,设计鼠源单克隆抗体的重链、轻链可变区基因引物,成功扩增获得FB1鼠源抗体的重链、轻链基因片段,大小为300～400bp。SOE-PCR将VH和VL由Linker拼接成FB1ScFv,大小为700～800bp。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,经过辅助噬菌体M13K07感染,构建得到的FBl噬菌体重组单链抗体库,抗体滴度约为2.1×1015cfu·mL-1。经过五轮免疫亲和富集和筛选,成功筛选得到5株可分泌FBl特异性噬菌体单链抗体的菌株。核苷酸序列鉴定显示该序列为717bp,编码239个氨基酸。在M13K07辅助噬菌体的辅助下,pCANTAB5E噬菌体质粒可产生含有FBl单链噬菌体基因组的噬菌体颗粒,并以融合的形式表达在噬菌体表面。在E.coli HB2151中,FB1ScFv基因在翻译过程中形成独立的抗体蛋白,以胞周间质形式形成可溶性蛋白。
试验Ⅲ赭曲霉毒素A单链抗体可变区基因克隆与噬茵体抗体蛋白表达
用OTA-BSA免疫BALB/c小鼠,免疫效价达到要求后,提取小鼠脾总RNA,反转录合成cDNA第一条链,应用PCR技术分别克隆OTA鼠源抗体VH和VL,并经过SOE-PCR,通过柔性多肽Linker接头,按VH-Linker-VL方式拼接成OTA ScFv片段。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,应用噬菌体展示技术,以OTA-OVA为固相包被抗原进行免疫亲和筛选,应用Phage-ELISA和ScFv-ELISA鉴定和筛选阳性噬菌体重组单链抗体。将阳性pCANTAB5E-OTA ScFv质粒转化入表达菌株E. coli HB2151中,经IPTG诱导,获得OTA特异性ScAb。结果显示,设计鼠源单克隆抗体的重链、轻链可变区基因引物,成功扩增获得OTA鼠源抗体的VH和VL,大小为300～400bp。SOE-PCR将VH和VL由Linker拼接成ScFv,大小为700～800bp。随后将ScFv基因重组入pCANTAB5E载体中,转化至E. coli TG1中,经过辅助噬菌体M13K07感染,构建得到的OTA噬菌体重组单链抗体库,抗体滴度约为2.65×1016cfu·mL-1。经过三轮免疫亲和富集和筛选,成功筛选得到3株可分泌OTA特异性噬菌体单链抗体的菌株。核苷酸序列鉴定显示该序列为738bp,共编码246个氨基酸。在pCANTAB5E噬菌体质粒中,OTA ScFv以胞周间质形式形成可溶性蛋白。
试验Ⅳ伏马菌素B1和赭曲霉毒素A单链抗体特性鉴定
应用蛋白免疫印迹(WB)和i-ELISA方法分别鉴定OTA与FB1两种真菌毒素ScAb的免疫活性。应用i-ELISA分别测定OTA与FB1ScAb的效价。应用间接竞争酶联免疫吸附试验(ci-ELISA)建立竞争抑制标准曲线。分别测定OTA和FB1ScAb的灵敏度,并测定OTA, FB1ScAb与参试毒素FB2、FB3、AFB1、ZEA和DON交叉反应率,以反映其特异性。结果显示,WB和i-ELISA均显示OTA与FB1ScAb具有良好的免疫活性。FB1-OVA抗原最佳工作浓度为5μg·mL-1, ScAb最佳稀释度为1:1600; OTA-OVA抗原最佳工作浓度为10μg-mL-1, ScAb最佳稀释度为1：800；FB1ScAb抗体滴度达到104; OTA ScAb抗体滴度达到103; ci-ELISA法绘制FB1竞争抑制曲线,FB1浓度为0.31～20μg-mL-1范围内曲线线性关系良好,线性方程为y=-16.663x+117.98(R2=0.9674), FB1ScAb对FB150%抑制浓度4.08μg·mL-1;同样方法绘制OTA竞争抑制曲线,OTA浓度为0.31～10μgmL-1范围内线性关系良好,线性方程为y=-16.075x+116.84(R2=0.9797),OTA ScAb对OTA的50%抑制浓度4.16μg·mL-1。FB1ScAb对FB1同种属的FB2、FB3的交叉反应抑制率分别为71.7%和87.6%,而与DON、AFB1、OTA和ZEA交叉反应抑制率较小,说明该FB1ScAb对FB1具有较好的特异性；OTA ScAb对FBi、DON、AFB1和ZEA交叉反应抑制率较小,说明该OTA ScAb对OTA具有较好的特异性。
Mycotoxins are a range of toxic secondary metabolites produced by various funguses and the contamination of grains and all kinds of agricultural products, which exist in nature widely. Mycotoxins have been a potential threat to human health and the development of animal husbandry that may not only reduce the quality of the products, but also lead to serious foodborne poisoning and cause people or animals to acute or chronic poisoning. Animal tests showeded that the animals that took in mycotoxins contaminated feed would have some pathological changes, such as immune suppression, tissue necrosis, liver and kidney damage, reproductive disorder, carcinogenesis and cacogenics and so on, and make direct or indirect impact on the health of humans and animals. Many kinds of mycotoxin exist in nature with complex toxicities. They have caused highly concerns over global food security owing to the widely pollution and toxicity. In order to prevent mycotoxins contaminated food and animal products from the human food chain, directly or indirectly, strengthening the detection of mycotoxins is essential. In recent years, the detection technologies for mycotoxins have been developed rapidly, such as the chromatographic detection technology mainly based on high-performance liquid chromatography (HPLC) and immunology detection technology based on enzyme-linked immunosorbent assay (ELISA). Though high sensitivity, chromatography analytical method has shortcomings, such as the high quality requirement for sample and complex pretreatment operation, which make it difficult to be applied in actual production extensively. Compared to this, immunological method, based on the antigen-antibody has been favored by people due to its easy operation and fast advantages. Now, some experts and scholars focus on the various aspects of optimization and improvement of ELISA method in order to improve the detection performance.
Genetically engineered antibody is the third generation of antibody that developed on the application of recombinant DNA technology and protein engineering technology and based on gene structure and function of the antibody. The significance of the genetically engineered antibody is to obtain the specific engineered protein with a series of operations, such as cutting, splicing and modification for antibody genes. Compared with polyclonal antibody and monoclonal antibody, genetically engineered antibody have its advantages, for example, short time prepare, simple operation, scale production and low cost. The Single-chain antibody (ScAb) is a member of genetic engineering antibody, which has been regarded as one direction of immunoassay technology development. As the improved product of traditional monoclonal antibody, ScAb has a better application prospect because of its stable and conserving easily. In this study, two kinds of ScAb against two common mycotoxins, ochratoxin A (OTA) and fumonisin B1(FB1) were obtained with molecular biology technique and phage display technique, so as to achieve a breakthrough both on theory and on technology, and would be the basic work for its immunoassay application and development.
Test I Synthesis and characterization of complete antigen for fumonisin B1and ochratoxin A
In order to obtain FB1and OTA antibody genes, FB1was coupled with carrier proteins ovalbumin (OVA) and bovine serum albumin (BSA) respectively, by one step glutaraldehyde method, while two complete antigens FB1-OVA and FB1-BSA were synthesized. OTA was coupled with OVA and BSA respectively to synthesized complete antigens OTA-BSA and OTA-OVA by carbodiimide method. Ultraviolet absorption spectroscopy (UV), gel electrophoresis, fourier transforms infrared spectroscopy (IR) and matrix-assisted laser absorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to determine the coupled effects and coupling ratios of the complete antigens, respectively. FB1-BSA and OTA-BSA were used to immunize BALB/c mice, and FB1-OVA and OTA-OVA were used as solid phase coating antigens, respectively. The indirect enzyme-linked immunosorbent assay (i-ELISA) was used to determine the titer of anti-serum from mice. The results showed that carrier proteins coupled with FB1and OTA successful, respectively. The coupling ratios of FB1-BSA, FB1-OVA, OTA-BSA and OTA-OVA were10:1,5:1,3:1and9:1by MALDI-TOF-MS method, respectively. The titers of anti-serum form the mice immunized FB1-BSA or OTA-BSA could be reached1:1.024×105determined by i-ELISA method, respectively. The complete antigens and the mice immunized could be used for the next test.
Test II The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against fumonisin B1
A total RNA was extracted from hybridoma cell line F3that secreted FB1monoclonal antibody. The first strand of cDNA was amplified by reverse transcription PCR (RT-PCR). The heavy chain and light chain variable region genes(VH and VL) were amplified by PCR used cDNA as the template, respectively. The FB1single chain antibody variable region gene fragment (ScFv) was splicing by overlap extension PCR (SOE-PCR) using a flexible the polypeptide (Gly4Ser)3as DNA linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into Escherichia coli (E. coli) host strain TG1and then rescued by M13K07helper phage. Biopanning and screening, FB1-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of FB1ScAb with IPTG. The results showed that FB1murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were348and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was717bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TG1, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.1×1015cfu·mL-1. After five times biopanning enrichment and screening, five strains that could secrete the FB1-specific phage single-chain antibody were selected successfully. Transformed cells were then infected with M13K07helper phage to rescue the phagemid with its ScFv gene insert. Recombinant phages which were produced contain a single-stranded DNA copy of the phagemid and antibody ScFv gene and display one or more copies of the recombinant antibody at their tips. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.
Test IH The cloning of single-chain antibody variable region gene and the expression of phage antibody protein against ochratoxin A
A total RNA was extracted from spleen cell of mice immunized by OTA-BSA. The first strand of cDNA was amplified by RT-PCR. The VH and VL were amplified by PCR used cDNA as the template, respectively. The OTA ScFv was splicing by SOE-PCR using a linker and according to VH-linker-VL direction. The ScFv genes were then assembled in plasmid vectors pCANTAB5E, transformed into E. coli TGI and then rescued by M13K07helper phage. Biopanning and screening, using OTA-OVA as solid phase coated antigen, was used to select for antigen-positive recombinant phage antibody based on phage display technology. The positive recombinant phage antibody was identified and screening using Phage-ELISA and ScFv-ELISA. Infection of E. coli HB2151by selected recombinant phage resulted in stable and efficient expression of OTA ScAb with IPTG. The results showed that OTA murine antibody heavy chain and light chain gene fragment were obtained using the primers, and the VH and VL were369and324bp, respectively. ScFv was splicing by SOE-PCR using VH, VL and linker, and ScFv was738bp. The ScFv genes were then assembled in pCANTAB5E and transformed into E. coli TGI, rescued by M13K07helper phage. A library of phage scFv antibody was constructed, and the antibody titer was approximately2.65×1016cfu·mL-1. After five times biopanning enrichment and screening, three strains that could secrete the OTA-specific phage single-chain antibody were selected successfully. When the E.coli HB2151was infected by recombinant phage, the soluble antibody was produced.
Test IV Characterization of single-chain antibody against fumonisin B1and ochratoxin A
The immune activities of OTA and FB1recombinant ScAb were detected by Western blotting (WB) and i-ELISA, respectively. The titers of OTA and FB1recombinant ScAb were detected by i-ELISA, respectively. The sensitivities and competitive inhibition standard curves of OTA and FB1recombinant ScAb were established by competitive indirect enzyme-linked immunosorbent assay (ci-ELISA), respectively. And the specificities and cross-relativities of OTA and FB1ScAb to test toxins were determined using FB2, FB3, AFB1, ZEA and DON, respectively. The results showed that good immune activities of OTA or FB1ScAb to antigen were proofed through WB and i-ELISA method. The best working concentration of FB1-OVA antigen was5μg·mL-1and the best dilution of FB1ScAb was1:1600. The FB1ScAb titers reached104. The FB1competitive inhibition curve was established by ci-ELISA method, the linear equation y=-16.663x+117.98(R2=0.9674) and50%inhibition concentration (IC50) for OTA was4.08μg·mL-1, ranging from0.31～20μg·mL-1. The cross-reactivity inhibition rates of FB1ScAb to FB2and FB3were71.7%and87.6%, and the significant cross-reactivity with DON, AFB1, OTA and ZEA. The FB1ScAb has the better activity, sensitivity and specificity. The best working concentration of OTA-OVA antigen was10μg·mL-1and the best dilution of OTA ScAb was1:800. The OTA ScAb titers reached103. The OTA competitive inhibition curve was established by ci-ELISA method, the linear equation y=16.075x+116.84(R2=0.9797) and IC5o for OTA was4.16μg·mL-1, ranging from0.31～10μg·mL-1. The OTA ScAb had little cross-reactivity with DON, AFB1and ZEA. The OTA ScAb has better activity, sensitivity and specificity.
引文
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