抗禽流感病毒H5N1分泌型IgA在CHO细胞中的表达及免疫学活性分析
发布时间：2018-07-24 16:09
【摘要】：禽流感(Avian influenza,AI)是由甲型流感病毒引起的一种以侵害呼吸系统为主的疾病,极大地危害人类健康和畜牧业生产,并严重影响国家的经济发展。禽流感病毒(Avian influenza virus,AIV)基因组极易发生变异,造成病毒的多型性,给禽流感防治带来很大难度。目前,还不清楚哪些特异突变可以使H5N1病毒能稳定地在人-人之间传播,但是一旦发生H5N1病毒突变导致对人的特异性,将会给人类造成重大灾难。 人们对于禽流感的预防与治疗主要集中在疫苗和抗病毒药物两种方法上,而疫苗从注射到产生保护性抗体需要较长时间。由于禽流感病毒对离子通道抑制剂、神经氨酸酶抑制剂等化学药物容易产生抗性,这将都会影响疾病的控制效果。近两年来有关禽流感被动免疫预防和治疗的研究是禽流感研究的热点之一。使用抗体制剂对禽流感进行被动免疫预防和治疗,可以在使用之后立刻产生防护效果,从而可以弥补疫苗和化学药物的不足。 禽流感是一种呼吸道传染病,感染途径主要是呼吸道和消化道。分泌型IgA(Secretory IgA,SIgA)抗体在这里具有重要作用,作为一种被动免疫制剂,在病毒感染发生的早期阶段给药,可有效地抵抗病毒的感染。另外,由于通过滴鼻/喷雾或口服/灌胃即可给药,这种非系统的、局部的用药方式不仅方便、安全,而且成本较低。因此,研究呼吸道或消化道分泌型IgA对禽流感病感染的阻断作用,具有潜在的应用价值。 分泌型IgA抗体在结构和功能上还具有独特的特点,与普通的IgG和IgA抗体分子相比,SIgA抗体有许多优良的特性。SIgA由2个IgA构成,故其与抗原的结合价比较高,具有较高的抗体活性。由于分泌片的存在,使SIgA可以抵抗酸和蛋白酶的降解,提高了SIgA的稳定性,有研究表明,SIgA分子可以在人体外分泌道存在四个月以上。此外,分泌片具有非特异性的病原体结合活性,从而使SIgA具有非特异的免疫保护作用;分泌片使SIgA整齐规则地排列在粘膜表面形成隔离保护层,可对病原微生物的入侵产生物理防护作用。 本研究利用基因工程手段,通过稳定转染CHO/dhfr-细胞系,构建了稳定表达分泌型IgA的单克隆细胞系,具体研究内容和结果如下: 1分泌片SC和IgJ基因真核表达质粒的构建 分泌型IgA相关基因(SC、IgJ基因、人IgA重链恒定区、人Kappa链恒定区)的克隆、抗H5N1单克隆抗体可变区基因(重链、轻链可变区)以及轻重链真核表达质粒的构建已由本实验室在前期工作中完成。基于优化的实验方案,我们又将SC、IgJ基因克隆到了含有Zeocin抗性基因的真核表达载体pcDNA4/HisA中,构建了SC和J链的真核表达载体pCDNA4-IgJ、pCDNA4-SC。 2抗H5N1-HA嵌合SIgA抗体在CHO细胞中的表达及免疫学特性分析 将抗H5N1抗体轻、重链真核表达质粒转染CHO/dhfr-细胞,筛选稳定分泌表达抗H5N1病毒的IgA抗体的单克隆细胞系,制备嵌合IgA单体抗体,并对其免疫学特性进行分析;然后在稳定表达IgA单体的中国仓鼠卵巢细胞(CHO)细胞系的基础上,共转染SC和J链真核表达质粒,利用Zeocin抗生素筛选分泌SIgA的单克隆细胞,通过ELISA和Western blotting分析了细胞培养上清中SIgA的表达情况,并证明表达的SIgA抗体具有完整的十聚体结构,而且ELISA实验也表明SIgA抗体与H5N1 HA抗原具有很好的结合能力。 3提高SIgA抗体表达量的相关研究 氨甲喋呤(MTX)是二氢叶酸还原酶(DHFR)抑制物,当培养基中存在MTX时,MTX可渗入细胞内与DHFR蛋白结合,使核苷酸的合成受阻,只有DHFR基因得到扩增的克隆才能存活。DHFR基因的扩增使其附近的DNA得到扩增,从而提高与之毗连的外源基因的表达水平。MTX加压程序是,先将待加压的细胞株传入细胞培养瓶中,将培养液换成含5nM MTX的加压培养基。当培养的细胞长到汇合度90%时,以1:4传代,连续传五代,然后换成25nM的MTX加压培养基,以此类推,最终加压浓度为125nM。MTX加压程过程中采用96孔板倍比稀释的方法筛选加压后的SIgA单克隆细胞系,获得表达量较高的细胞株。 4分泌型IgA的发酵培养及纯化 将SIgA单克隆细胞无血清培养驯化2个月,采用CD CHO培养基(含一定浓度的MTX),逐渐降低DMEM和透析血清浓度,直至其完全适应CD CHO生长;悬浮培养2-4周,培养基仍为CD CHO(含加压浓度的MTX)。收集细胞培养上清1L左右利用Protein-L的亲和层析柱进行纯化。 5分泌片SC单克隆抗体的制备 在SIgA抗体检测过程中,需要大量SC单抗,我们首先构建了原核表达载体pQE80L-SC,然后进行SC蛋白的诱导表达,SC蛋白经过纯化回收免疫小鼠,制备了2株单克隆抗体,并利用这些抗体建立了SIgA的快速诊断检测方法。
[Abstract]:Avian influenza (AI) is a disease caused by influenza A virus, which is mainly caused by the respiratory system, which greatly endangers human health and livestock production, and seriously affects the economic development of the country. The genome of the avian influenza virus (Avian influenza virus, AIV) is susceptible to variation of the genome, resulting in the polymorphism of the virus and the prevention of avian influenza. Treatment brings great difficulty. At present, it is not clear which specific mutations can enable H5N1 virus to spread steadily between human beings, but once the mutation of the H5N1 virus causes the human specificity, it will cause a major disaster for human beings.
The prevention and treatment of avian influenza is mainly focused on two methods of vaccine and antiviral drugs, and vaccines from injection to producing protective antibodies take a long time. Because of avian influenza virus, chemical drugs such as ion channel inhibitors and neuraminidase inhibitors are easy to produce resistance, which will affect the control effect of the disease. The research on the passive immunization and treatment of avian influenza in the last two years is one of the hotspots in the research of avian influenza. The use of antibody preparation to prevent and treat avian influenza can produce protective effects immediately after use, which can make up for the shortage of vaccines and chemicals.
Avian influenza is a kind of respiratory infectious disease, the main way of infection is respiratory and digestive tract. The antibody of secretory IgA (Secretory IgA, SIgA) plays an important role here. As a passive immune agent, it is administered at the early stage of the virus infection and can effectively resist the infection of the disease. In addition, it is due to nasal drip / spray or oral / irrigation. The stomach can be given. This non systematic and local use is not only convenient, safe, but also low cost. Therefore, it is of potential application value to study the blocking effect of respiratory tract or digestive tract secretory IgA on avian influenza infection.
Secretory IgA antibody also has unique characteristics in structure and function. Compared with common IgG and IgA antibody, SIgA antibody has many excellent properties,.SIgA is composed of 2 IgA, so its binding valence to antigen is higher and has higher antibody activity. As the secretory fragment is stored, the SIgA can resist the degradation of acid and protease. The stability of SIgA is higher. Some studies have shown that the SIgA molecule can exist in the exocrine tract for more than four months. In addition, the secretory tablet has non specific pathogen binding activity, which makes SIgA have a non specific protective effect. The secretory slice makes SIgA orderly and regularly arranged on the surface of the mucous membrane to form a isolation protective layer and can be microbiological to the pathogen. The invasion of objects has the effect of physical protection.
In this study, a monoclonal cell line for stable expression of secretory IgA was constructed by means of genetic engineering and stable transfection of CHO/dhfr- cell lines. The specific contents and results were as follows:
Construction of eukaryotic expression plasmid of SC and IgJ genes in secretory slices
The cloning of secretory IgA related genes (SC, IgJ gene, human IgA heavy chain constant region, human Kappa chain constant region), the construction of anti H5N1 monoclonal antibody variable region gene (heavy chain, light chain variable region) and heavy chain eukaryotic expression plasmid have been completed in the previous work in our laboratory. Based on the optimized experimental scheme, we cloned the SC, IgJ gene. Eukaryotic expression vectors pCDNA4-IgJ and pCDNA4-SC with SC and J chains were constructed in the eukaryotic expression vector pcDNA4/HisA containing Zeocin resistance gene.
Expression and immunological characteristics of anti-H5N1-HA chimeric SIgA antibody in CHO cells
The anti H5N1 antibody light and heavy chain eukaryotic expression plasmid transfected to CHO/dhfr- cells, the monoclonal cell lines that secrete the IgA antibody that express the anti H5N1 virus were screened, and the chimeric IgA monomer antibody was prepared and the immunological characteristics were analyzed. Then, on the basis of the stable expression of IgA monomer in Chinese silo ovarian cell line (CHO) cell line, SC was co transfected with SC. And J chain eukaryotic expression plasmid, using Zeocin antibiotics to screen the monoclonal cells that secrete SIgA, and analyze the expression of SIgA in cell culture supernatant by ELISA and Western blotting, and prove that the expressed SIgA antibody has a complete ten polymer structure, and ELISA experiment also shows that the SIgA antibody has a good binding energy to H5N1 HA antigen. Power.
3 Correlation Study on improving the expression of SIgA antibody
Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor. When MTX exists in the medium, MTX can infiltrate into the cell and bind to the DHFR protein to make the synthesis of the nucleotides blocked. Only the DHFR gene is amplified to survive the amplification of the.DHFR gene to amplify the DNA in the vicinity of the.DHFR gene, thus improving the table of the foreign genes contiguous to them. The level.MTX pressurization program is first to afferent the pressurized cell strain into a cell culture bottle and convert the culture medium into a pressurized medium containing 5nM MTX. When the cultured cells grow to the confluence of 90%, the cultured cells are passaged by 1:4 for five generations and then converted into 25nM MTX pressurized medium, so that the final pressure concentration is extracted during the 125nM.MTX compression process. The SIgA monoclonal cell lines were screened by 96-well plate dilution method and the high expression cell lines were obtained.
Fermentation and purification of 4 secretory IgA
The SIgA monoclonal cell was cultured for 2 months without serum-free culture, and the CD CHO medium (containing a certain concentration of MTX) was used to gradually reduce the concentration of DMEM and dialysis serum, until it was fully adapted to CD CHO growth. The medium was still CD CHO (MTX) with CD CHO (containing the pressure concentration) for 2-4 weeks. Purify.
Preparation of monoclonal antibodies against 5 secretory SC
In the process of SIgA antibody detection, a large number of SC monoclonal antibodies are needed. We first constructed the prokaryotic expression vector pQE80L-SC, and then induced the expression of SC protein. The SC protein was purified and recovered to be immune to mice. 2 monoclonal antibodies were prepared, and the rapid diagnosis and detection method of SIgA was established by using these antibodies.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2141913
[Abstract]:Avian influenza (AI) is a disease caused by influenza A virus, which is mainly caused by the respiratory system, which greatly endangers human health and livestock production, and seriously affects the economic development of the country. The genome of the avian influenza virus (Avian influenza virus, AIV) is susceptible to variation of the genome, resulting in the polymorphism of the virus and the prevention of avian influenza. Treatment brings great difficulty. At present, it is not clear which specific mutations can enable H5N1 virus to spread steadily between human beings, but once the mutation of the H5N1 virus causes the human specificity, it will cause a major disaster for human beings.
The prevention and treatment of avian influenza is mainly focused on two methods of vaccine and antiviral drugs, and vaccines from injection to producing protective antibodies take a long time. Because of avian influenza virus, chemical drugs such as ion channel inhibitors and neuraminidase inhibitors are easy to produce resistance, which will affect the control effect of the disease. The research on the passive immunization and treatment of avian influenza in the last two years is one of the hotspots in the research of avian influenza. The use of antibody preparation to prevent and treat avian influenza can produce protective effects immediately after use, which can make up for the shortage of vaccines and chemicals.
Avian influenza is a kind of respiratory infectious disease, the main way of infection is respiratory and digestive tract. The antibody of secretory IgA (Secretory IgA, SIgA) plays an important role here. As a passive immune agent, it is administered at the early stage of the virus infection and can effectively resist the infection of the disease. In addition, it is due to nasal drip / spray or oral / irrigation. The stomach can be given. This non systematic and local use is not only convenient, safe, but also low cost. Therefore, it is of potential application value to study the blocking effect of respiratory tract or digestive tract secretory IgA on avian influenza infection.
Secretory IgA antibody also has unique characteristics in structure and function. Compared with common IgG and IgA antibody, SIgA antibody has many excellent properties,.SIgA is composed of 2 IgA, so its binding valence to antigen is higher and has higher antibody activity. As the secretory fragment is stored, the SIgA can resist the degradation of acid and protease. The stability of SIgA is higher. Some studies have shown that the SIgA molecule can exist in the exocrine tract for more than four months. In addition, the secretory tablet has non specific pathogen binding activity, which makes SIgA have a non specific protective effect. The secretory slice makes SIgA orderly and regularly arranged on the surface of the mucous membrane to form a isolation protective layer and can be microbiological to the pathogen. The invasion of objects has the effect of physical protection.
In this study, a monoclonal cell line for stable expression of secretory IgA was constructed by means of genetic engineering and stable transfection of CHO/dhfr- cell lines. The specific contents and results were as follows:
Construction of eukaryotic expression plasmid of SC and IgJ genes in secretory slices
The cloning of secretory IgA related genes (SC, IgJ gene, human IgA heavy chain constant region, human Kappa chain constant region), the construction of anti H5N1 monoclonal antibody variable region gene (heavy chain, light chain variable region) and heavy chain eukaryotic expression plasmid have been completed in the previous work in our laboratory. Based on the optimized experimental scheme, we cloned the SC, IgJ gene. Eukaryotic expression vectors pCDNA4-IgJ and pCDNA4-SC with SC and J chains were constructed in the eukaryotic expression vector pcDNA4/HisA containing Zeocin resistance gene.
Expression and immunological characteristics of anti-H5N1-HA chimeric SIgA antibody in CHO cells
The anti H5N1 antibody light and heavy chain eukaryotic expression plasmid transfected to CHO/dhfr- cells, the monoclonal cell lines that secrete the IgA antibody that express the anti H5N1 virus were screened, and the chimeric IgA monomer antibody was prepared and the immunological characteristics were analyzed. Then, on the basis of the stable expression of IgA monomer in Chinese silo ovarian cell line (CHO) cell line, SC was co transfected with SC. And J chain eukaryotic expression plasmid, using Zeocin antibiotics to screen the monoclonal cells that secrete SIgA, and analyze the expression of SIgA in cell culture supernatant by ELISA and Western blotting, and prove that the expressed SIgA antibody has a complete ten polymer structure, and ELISA experiment also shows that the SIgA antibody has a good binding energy to H5N1 HA antigen. Power.
3 Correlation Study on improving the expression of SIgA antibody
Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor. When MTX exists in the medium, MTX can infiltrate into the cell and bind to the DHFR protein to make the synthesis of the nucleotides blocked. Only the DHFR gene is amplified to survive the amplification of the.DHFR gene to amplify the DNA in the vicinity of the.DHFR gene, thus improving the table of the foreign genes contiguous to them. The level.MTX pressurization program is first to afferent the pressurized cell strain into a cell culture bottle and convert the culture medium into a pressurized medium containing 5nM MTX. When the cultured cells grow to the confluence of 90%, the cultured cells are passaged by 1:4 for five generations and then converted into 25nM MTX pressurized medium, so that the final pressure concentration is extracted during the 125nM.MTX compression process. The SIgA monoclonal cell lines were screened by 96-well plate dilution method and the high expression cell lines were obtained.
Fermentation and purification of 4 secretory IgA
The SIgA monoclonal cell was cultured for 2 months without serum-free culture, and the CD CHO medium (containing a certain concentration of MTX) was used to gradually reduce the concentration of DMEM and dialysis serum, until it was fully adapted to CD CHO growth. The medium was still CD CHO (MTX) with CD CHO (containing the pressure concentration) for 2-4 weeks. Purify.
Preparation of monoclonal antibodies against 5 secretory SC
In the process of SIgA antibody detection, a large number of SC monoclonal antibodies are needed. We first constructed the prokaryotic expression vector pQE80L-SC, and then induced the expression of SC protein. The SC protein was purified and recovered to be immune to mice. 2 monoclonal antibodies were prepared, and the rapid diagnosis and detection method of SIgA was established by using these antibodies.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
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