GICA一步法检测HIV和TP诊断试剂的研究
摘要
本研究采用胶体金免疫层析技术(GICA)研制一滴血一次实验检测两种疾病,制备艾滋病毒(HIV)抗体和梅毒(TP)抗体一步双联免疫胶体金快速诊断试剂盒,经过实验条件优选,使此检测方法灵敏、精准、特异,操作简便、快速,提高技术含量,降低检测成本,便于流动监测,使其适用于现场实地监测,达到及时准确地对HIV和TP进行筛查、监测的目的。
本研究对HIV和TP的抗原片断进行了优化选择,通过计算机分析同源序列确定了新的抗原位点。成功构建了由HIV-1/2和TP外膜蛋白多个抗原位点串联基因片段的表达载体pRSETB-env和pRSETB-tp,在原核表达载体系统中对HIV-1gp41/gp120、HIV-2gp125/gp36及TpN15、TpN17、TpN44.5、TpN47多个抗原位点同源序列合成基因进行表达、纯化并鉴定其活性,采用胶体金免疫层析技术(GICA)双联HIV和TP抗原,一步同时检测HIV和TP抗体。
结果显示目的基因在BL21中有较高表达率,纯化后表达蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)可见与设计相对分子质量相符的蛋白带。免疫印迹、ELISA试验显示pRSETB-env质粒的HIV-1/2表达蛋白与HIV-1及HIV-2患者血清都能较好的结合,与其他患者血清无交叉反应;pRSETB-tp质粒的TP表达蛋白与TP患者血清都能较好的结合,与其他患者血清无交叉反应。
成功构建了一步法检测HIV和TP的胶体金诊断试剂,胶体金试纸能高效的检测HIV和TP患者血清,具有较高的特异性和敏感性。为一步、同时、快速检测HIV和TP抗体试剂盒的研制和开发提供了重要的研究成果,具有广阔的应用前景。
HIV, known as the human immunodeficiency virus, is the pathogens of Human acquired immunodeficiency syndrome. After HIV invaded into the human body, it can destroy the body's immune function and induce a series of incurable infections and tumors, and can result in death. HIV spread rapidly in human and can cause high mortality rate. HIV is a RNA virus with envelope, it belongs to Retroviral Branch of the lentivirus subfamily. The shape of HIV is a spherical pocket and it's diameter is about 150 nm. HIV has genetic diversity, about more than 40 kinds of different antigen and genetic subtypes has been found today. Detection of HIV antibodies in the blood is the most commonly used methods for detection of HIV infection in laboratory.
According to the difference of genetics and serological characteristic, HIV can be divided into HIV-1 and HIV-2, and now all the world countries have found two kinds of virus. The endemic trend of HIV-1 / 2 co-infection is increasing in some countries with HIV-1 and/or HIV-2 infection. Such as the infection rate of Portugal is1.4%, Guinea-Bissau is 0.7%, Senegal is 0.4% and India is>2%. However, because of the lack of sensitivity and specificity of the HIV-2 antibody detection kit, the true infection rate may be far greater than the above statistics.
TP, known as Treponema Pallidun, is the pathogens of syphilis. Once infected by TP, it can cause genital mutilation and any organs and tissues can be endangered, the clinical manifestations has a widely variety. Human is the only source of Syphilis infection. TP can be transmitted to the fetus through the placenta, and 95% of direct infections are from sexual intercourse. TP is a small and thin microorganisms in a spiral structure, the length of it is about 5-20 nm, the diameter of it is <0.2 nm, and it has a 6-12 spiral. After TP invaded into the human body 48 hours, the specific antibodies can be produced in the blood. Serological testing often the spiral surface-specific antigen, type-specific antigen in a spiral or helical and phospholipids antigen formatted by host organization often are the target antigens for specific antibodies detection in serological testing.
Gold immunochromatography assay (GICA) is an organic combination of solid-state tag immune detection technology with colloidal gold labeling technology, chromatography analysis technology and other methods. The principle of GICA is: Under the conditions of reducing agent, the acid chloride (HAuCl4) will polymerize in certain particle size with a negative hydrophobic plastic solution, named colloidal gold. Gold-tag technology is the process of protein coated on surface of gold particles though absorption by the negative charge of protein and positive charge of gold particles. The main principle of Immunogold labeling technology is the utility of gold particles with high electron density characteristics. Under the microscope, gold can be showed in brown particles. When these markers gathered largely in the appropriate allocation, they can be visible in red or pink. Thus, GICA can by used in semi-quantitative or qualitative methods for the rapid immunized detection.
The purpose of this study is expression, purification and activity identification of HIV-1gp41/gp120, HIV-2gp125/gp36 and TpN15, TpN17, TpN44.5, TpN47 multiple antigen in prokaryotic expression vector system. Use of Gold immunochromatography assay (GICA) and TP-HIV antigens, we will develop one-step detection methods for TP and HIV antibodies testing.
Use of a series of synthetic genes for three antigen sites of HIV-1gp41, two antigen sites of HIV-1gp120, three antigen sites of HIV-2gp125, one antigen site of HIV-2gp36, two antigen sites of TpN15, one antigen site of TPN17, one antigen site of TpN44.5 and one antigen site of TpN47, we cloned these genes into prokaryotic expression vector pRSETB to construct recombinant expression plasmid pRSETB-env and pRSETB-tp. The results showed that the aimed proteins could express highly in E. coli BL21 (DE3) cells induced by IPTG. The aimed proteins were purified by metal ions affinity chromatography and refolded by gradually reduce the concentration of urea, the purified proteins were identified by ELISA and Western blot. The aimed proteins were coated in colloidal gold solution, and colloidal gold test papers were prepared for testing HIV and TP antibodies in patient’s serum.
Prokaryotic expression and eukaryotic expression has its own advantages and disadvantages. The Prokaryotic expression has the advantages of low-cost, high production and easy manufacture. But because of the lack of the necessary modification, it’s activity is often less than natural protein. Eukaryotic expression of the protein in a follow-up in the course of the modification, such as glycosylation, and so on, which makes the expression of eukaryotic protein in the biological activity closer to natural areas. However, due to high costs of eukaryotic expression, access to adequate protein activity cycle is too long, so many proteins in the study, the prokaryotic expression still occupies very heavy proportion. At the same time, many studies have found that the prokaryotic expression of the HIV antigen and TP antigen still have a very high sensitivity in the detection, so we constructed 10 types of prokaryotic expression vector for HIV-1 / 2 and TP antigens, and highly expressed in E. coli BL21 (DE3) cells for activity proteins.
At present, there are a great variety of prokaryotic expression vectors. In design of these prokaryotic expression vectors, most strong promoter such as T7 promoter for the introduction of a large number of protein expression are used, and the choice for protein purification tags such as the GST and His tags are used, as well as appropriate resistance genes such as Amp and Kan genes for clone selection. Currently, the prokaryotic expression vector such as pET series and pRSET series are widely used in laboratory research. pRSET series prokaryotic expression vector is one of classic expression vector of INVITROGEN company, it contains the T7 promoter suitable for protein expression, and His tag for protein purification, and Amp resistance gene for clone selection as well as multiple clone sites for foreign gene insertion. So, we used pRSET-B expression vector for foreign genes expression in this paper.
Through searching in Genebank database for full-length genes of HIV-1/2 virus and Tp spiral, and comparison with relative literature, we determined major genes sequence of dominant antigens of HIV-1/2 virus and Tp spiral. All of the genes connected by suitable linker gene, and 1041bp of HIV gene and 669bp of TP gene were acquired by artificial gene synthesis methods. Because the original gene sequences are from virus and spiral, which are not suitable for expression in the E. coli, we did do codon usage for aimed genes highly expression in E.coli.cells. And then to HIV and TP gene as a template, we design a series of specific primers for each aimed genes, and primers were introduced in the BamH
Ⅰ, HindⅢand EcoRⅠrestriction site. For follow-up protein identification, the myc tag sequence is also introduced in antisense primers, and then the gene is amplified by high-fidelity Platinum Taq enzyme. Based on the molecular biology methods, 10 interest genes were inserted in prokaryotic expression vector through sub-clone and clone.
In this study we successfully constructed prokaryotic expression vector pRSETB-env and pRSETB-tp, and aimed genes could be expressed highly in E. coli BL21 (DE3) cells by SDS-PAGE and the molecular weight of proteins was consisted with expectation. The results of western blot and ELISA showed that HIV-1/2 protein expressed by pRSETB-env plasmid could hybrid with serum of HIV-1 and HIV-2 patients and without cross reaction for other serum; TP protein expressed by pRSETB-tp plasmid could hybrid with serum of TP patients and without cross reaction for other serum. This result confirmed the homologous sequence of the expression antigen gene is highly sensitive and specificity.
we successfully constructed a one-step detection of HIV and TP colloidal gold diagnostic reagents, it could hybrid with serum of HIV-1,HIV-2 and TP patients and without cross reaction for other serum. This result confirmed, a one-step detection of HIV and TP colloidal gold diagnostic reagents, the test is highly sensitive and specificity. This study provide an important experimental basis for developing one-step rapid diagnostic kits for HIV and TP antibodies, so this study has a wider application.
本研究对HIV和TP的抗原片断进行了优化选择,通过计算机分析同源序列确定了新的抗原位点。成功构建了由HIV-1/2和TP外膜蛋白多个抗原位点串联基因片段的表达载体pRSETB-env和pRSETB-tp,在原核表达载体系统中对HIV-1gp41/gp120、HIV-2gp125/gp36及TpN15、TpN17、TpN44.5、TpN47多个抗原位点同源序列合成基因进行表达、纯化并鉴定其活性,采用胶体金免疫层析技术(GICA)双联HIV和TP抗原,一步同时检测HIV和TP抗体。
结果显示目的基因在BL21中有较高表达率,纯化后表达蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)可见与设计相对分子质量相符的蛋白带。免疫印迹、ELISA试验显示pRSETB-env质粒的HIV-1/2表达蛋白与HIV-1及HIV-2患者血清都能较好的结合,与其他患者血清无交叉反应;pRSETB-tp质粒的TP表达蛋白与TP患者血清都能较好的结合,与其他患者血清无交叉反应。
成功构建了一步法检测HIV和TP的胶体金诊断试剂,胶体金试纸能高效的检测HIV和TP患者血清,具有较高的特异性和敏感性。为一步、同时、快速检测HIV和TP抗体试剂盒的研制和开发提供了重要的研究成果,具有广阔的应用前景。
HIV, known as the human immunodeficiency virus, is the pathogens of Human acquired immunodeficiency syndrome. After HIV invaded into the human body, it can destroy the body's immune function and induce a series of incurable infections and tumors, and can result in death. HIV spread rapidly in human and can cause high mortality rate. HIV is a RNA virus with envelope, it belongs to Retroviral Branch of the lentivirus subfamily. The shape of HIV is a spherical pocket and it's diameter is about 150 nm. HIV has genetic diversity, about more than 40 kinds of different antigen and genetic subtypes has been found today. Detection of HIV antibodies in the blood is the most commonly used methods for detection of HIV infection in laboratory.
According to the difference of genetics and serological characteristic, HIV can be divided into HIV-1 and HIV-2, and now all the world countries have found two kinds of virus. The endemic trend of HIV-1 / 2 co-infection is increasing in some countries with HIV-1 and/or HIV-2 infection. Such as the infection rate of Portugal is1.4%, Guinea-Bissau is 0.7%, Senegal is 0.4% and India is>2%. However, because of the lack of sensitivity and specificity of the HIV-2 antibody detection kit, the true infection rate may be far greater than the above statistics.
TP, known as Treponema Pallidun, is the pathogens of syphilis. Once infected by TP, it can cause genital mutilation and any organs and tissues can be endangered, the clinical manifestations has a widely variety. Human is the only source of Syphilis infection. TP can be transmitted to the fetus through the placenta, and 95% of direct infections are from sexual intercourse. TP is a small and thin microorganisms in a spiral structure, the length of it is about 5-20 nm, the diameter of it is <0.2 nm, and it has a 6-12 spiral. After TP invaded into the human body 48 hours, the specific antibodies can be produced in the blood. Serological testing often the spiral surface-specific antigen, type-specific antigen in a spiral or helical and phospholipids antigen formatted by host organization often are the target antigens for specific antibodies detection in serological testing.
Gold immunochromatography assay (GICA) is an organic combination of solid-state tag immune detection technology with colloidal gold labeling technology, chromatography analysis technology and other methods. The principle of GICA is: Under the conditions of reducing agent, the acid chloride (HAuCl4) will polymerize in certain particle size with a negative hydrophobic plastic solution, named colloidal gold. Gold-tag technology is the process of protein coated on surface of gold particles though absorption by the negative charge of protein and positive charge of gold particles. The main principle of Immunogold labeling technology is the utility of gold particles with high electron density characteristics. Under the microscope, gold can be showed in brown particles. When these markers gathered largely in the appropriate allocation, they can be visible in red or pink. Thus, GICA can by used in semi-quantitative or qualitative methods for the rapid immunized detection.
The purpose of this study is expression, purification and activity identification of HIV-1gp41/gp120, HIV-2gp125/gp36 and TpN15, TpN17, TpN44.5, TpN47 multiple antigen in prokaryotic expression vector system. Use of Gold immunochromatography assay (GICA) and TP-HIV antigens, we will develop one-step detection methods for TP and HIV antibodies testing.
Use of a series of synthetic genes for three antigen sites of HIV-1gp41, two antigen sites of HIV-1gp120, three antigen sites of HIV-2gp125, one antigen site of HIV-2gp36, two antigen sites of TpN15, one antigen site of TPN17, one antigen site of TpN44.5 and one antigen site of TpN47, we cloned these genes into prokaryotic expression vector pRSETB to construct recombinant expression plasmid pRSETB-env and pRSETB-tp. The results showed that the aimed proteins could express highly in E. coli BL21 (DE3) cells induced by IPTG. The aimed proteins were purified by metal ions affinity chromatography and refolded by gradually reduce the concentration of urea, the purified proteins were identified by ELISA and Western blot. The aimed proteins were coated in colloidal gold solution, and colloidal gold test papers were prepared for testing HIV and TP antibodies in patient’s serum.
Prokaryotic expression and eukaryotic expression has its own advantages and disadvantages. The Prokaryotic expression has the advantages of low-cost, high production and easy manufacture. But because of the lack of the necessary modification, it’s activity is often less than natural protein. Eukaryotic expression of the protein in a follow-up in the course of the modification, such as glycosylation, and so on, which makes the expression of eukaryotic protein in the biological activity closer to natural areas. However, due to high costs of eukaryotic expression, access to adequate protein activity cycle is too long, so many proteins in the study, the prokaryotic expression still occupies very heavy proportion. At the same time, many studies have found that the prokaryotic expression of the HIV antigen and TP antigen still have a very high sensitivity in the detection, so we constructed 10 types of prokaryotic expression vector for HIV-1 / 2 and TP antigens, and highly expressed in E. coli BL21 (DE3) cells for activity proteins.
At present, there are a great variety of prokaryotic expression vectors. In design of these prokaryotic expression vectors, most strong promoter such as T7 promoter for the introduction of a large number of protein expression are used, and the choice for protein purification tags such as the GST and His tags are used, as well as appropriate resistance genes such as Amp and Kan genes for clone selection. Currently, the prokaryotic expression vector such as pET series and pRSET series are widely used in laboratory research. pRSET series prokaryotic expression vector is one of classic expression vector of INVITROGEN company, it contains the T7 promoter suitable for protein expression, and His tag for protein purification, and Amp resistance gene for clone selection as well as multiple clone sites for foreign gene insertion. So, we used pRSET-B expression vector for foreign genes expression in this paper.
Through searching in Genebank database for full-length genes of HIV-1/2 virus and Tp spiral, and comparison with relative literature, we determined major genes sequence of dominant antigens of HIV-1/2 virus and Tp spiral. All of the genes connected by suitable linker gene, and 1041bp of HIV gene and 669bp of TP gene were acquired by artificial gene synthesis methods. Because the original gene sequences are from virus and spiral, which are not suitable for expression in the E. coli, we did do codon usage for aimed genes highly expression in E.coli.cells. And then to HIV and TP gene as a template, we design a series of specific primers for each aimed genes, and primers were introduced in the BamH
Ⅰ, HindⅢand EcoRⅠrestriction site. For follow-up protein identification, the myc tag sequence is also introduced in antisense primers, and then the gene is amplified by high-fidelity Platinum Taq enzyme. Based on the molecular biology methods, 10 interest genes were inserted in prokaryotic expression vector through sub-clone and clone.
In this study we successfully constructed prokaryotic expression vector pRSETB-env and pRSETB-tp, and aimed genes could be expressed highly in E. coli BL21 (DE3) cells by SDS-PAGE and the molecular weight of proteins was consisted with expectation. The results of western blot and ELISA showed that HIV-1/2 protein expressed by pRSETB-env plasmid could hybrid with serum of HIV-1 and HIV-2 patients and without cross reaction for other serum; TP protein expressed by pRSETB-tp plasmid could hybrid with serum of TP patients and without cross reaction for other serum. This result confirmed the homologous sequence of the expression antigen gene is highly sensitive and specificity.
we successfully constructed a one-step detection of HIV and TP colloidal gold diagnostic reagents, it could hybrid with serum of HIV-1,HIV-2 and TP patients and without cross reaction for other serum. This result confirmed, a one-step detection of HIV and TP colloidal gold diagnostic reagents, the test is highly sensitive and specificity. This study provide an important experimental basis for developing one-step rapid diagnostic kits for HIV and TP antibodies, so this study has a wider application.
引文
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