抗禽流感病毒H5N1分泌型IgA在CHO細(xì)胞中的表達(dá)及免疫學(xué)活性分析
發(fā)布時(shí)間：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種以侵害呼吸系統(tǒng)為主的疾病,極大地危害人類健康和畜牧業(yè)生產(chǎn),并嚴(yán)重影響國家的經(jīng)濟(jì)發(fā)展。禽流感病毒(Avian influenza virus,AIV)基因組極易發(fā)生變異,造成病毒的多型性,給禽流感防治帶來很大難度。目前,還不清楚哪些特異突變可以使H5N1病毒能穩(wěn)定地在人-人之間傳播,但是一旦發(fā)生H5N1病毒突變導(dǎo)致對人的特異性,將會給人類造成重大災(zāi)難。 人們對于禽流感的預(yù)防與治療主要集中在疫苗和抗病毒藥物兩種方法上,而疫苗從注射到產(chǎn)生保護(hù)性抗體需要較長時(shí)間。由于禽流感病毒對離子通道抑制劑、神經(jīng)氨酸酶抑制劑等化學(xué)藥物容易產(chǎn)生抗性,這將都會影響疾病的控制效果。近兩年來有關(guān)禽流感被動免疫預(yù)防和治療的研究是禽流感研究的熱點(diǎn)之一。使用抗體制劑對禽流感進(jìn)行被動免疫預(yù)防和治療,可以在使用之后立刻產(chǎn)生防護(hù)效果,從而可以彌補(bǔ)疫苗和化學(xué)藥物的不足。 禽流感是一種呼吸道傳染病,感染途徑主要是呼吸道和消化道。分泌型IgA(Secretory IgA,SIgA)抗體在這里具有重要作用,作為一種被動免疫制劑,在病毒感染發(fā)生的早期階段給藥,可有效地抵抗病毒的感染。另外,由于通過滴鼻/噴霧或口服/灌胃即可給藥,這種非系統(tǒng)的、局部的用藥方式不僅方便、安全,而且成本較低。因此,研究呼吸道或消化道分泌型IgA對禽流感病感染的阻斷作用,具有潛在的應(yīng)用價(jià)值。 分泌型IgA抗體在結(jié)構(gòu)和功能上還具有獨(dú)特的特點(diǎn),與普通的IgG和IgA抗體分子相比,SIgA抗體有許多優(yōu)良的特性。SIgA由2個(gè)IgA構(gòu)成,故其與抗原的結(jié)合價(jià)比較高,具有較高的抗體活性。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的穩(wěn)定性,有研究表明,SIgA分子可以在人體外分泌道存在四個(gè)月以上。此外,分泌片具有非特異性的病原體結(jié)合活性,從而使SIgA具有非特異的免疫保護(hù)作用;分泌片使SIgA整齊規(guī)則地排列在粘膜表面形成隔離保護(hù)層,可對病原微生物的入侵產(chǎn)生物理防護(hù)作用。 本研究利用基因工程手段,通過穩(wěn)定轉(zhuǎn)染CHO/dhfr-細(xì)胞系,構(gòu)建了穩(wěn)定表達(dá)分泌型IgA的單克隆細(xì)胞系,具體研究內(nèi)容和結(jié)果如下: 1分泌片SC和IgJ基因真核表達(dá)質(zhì)粒的構(gòu)建 分泌型IgA相關(guān)基因(SC、IgJ基因、人IgA重鏈恒定區(qū)、人Kappa鏈恒定區(qū))的克隆、抗H5N1單克隆抗體可變區(qū)基因(重鏈、輕鏈可變區(qū))以及輕重鏈真核表達(dá)質(zhì)粒的構(gòu)建已由本實(shí)驗(yàn)室在前期工作中完成�；趦�(yōu)化的實(shí)驗(yàn)方案,我們又將SC、IgJ基因克隆到了含有Zeocin抗性基因的真核表達(dá)載體pcDNA4/HisA中,構(gòu)建了SC和J鏈的真核表達(dá)載體pCDNA4-IgJ、pCDNA4-SC。 2抗H5N1-HA嵌合SIgA抗體在CHO細(xì)胞中的表達(dá)及免疫學(xué)特性分析 將抗H5N1抗體輕、重鏈真核表達(dá)質(zhì)粒轉(zhuǎn)染CHO/dhfr-細(xì)胞,篩選穩(wěn)定分泌表達(dá)抗H5N1病毒的IgA抗體的單克隆細(xì)胞系,制備嵌合IgA單體抗體,并對其免疫學(xué)特性進(jìn)行分析;然后在穩(wěn)定表達(dá)IgA單體的中國倉鼠卵巢細(xì)胞(CHO)細(xì)胞系的基礎(chǔ)上,共轉(zhuǎn)染SC和J鏈真核表達(dá)質(zhì)粒,利用Zeocin抗生素篩選分泌SIgA的單克隆細(xì)胞,通過ELISA和Western blotting分析了細(xì)胞培養(yǎng)上清中SIgA的表達(dá)情況,并證明表達(dá)的SIgA抗體具有完整的十聚體結(jié)構(gòu),而且ELISA實(shí)驗(yàn)也表明SIgA抗體與H5N1 HA抗原具有很好的結(jié)合能力。 3提高SIgA抗體表達(dá)量的相關(guān)研究 氨甲喋呤(MTX)是二氫葉酸還原酶(DHFR)抑制物,當(dāng)培養(yǎng)基中存在MTX時(shí),MTX可滲入細(xì)胞內(nèi)與DHFR蛋白結(jié)合,使核苷酸的合成受阻,只有DHFR基因得到擴(kuò)增的克隆才能存活。DHFR基因的擴(kuò)增使其附近的DNA得到擴(kuò)增,從而提高與之毗連的外源基因的表達(dá)水平。MTX加壓程序是,先將待加壓的細(xì)胞株傳入細(xì)胞培養(yǎng)瓶中,將培養(yǎng)液換成含5nM MTX的加壓培養(yǎng)基。當(dāng)培養(yǎng)的細(xì)胞長到匯合度90%時(shí),以1:4傳代,連續(xù)傳五代,然后換成25nM的MTX加壓培養(yǎng)基,以此類推,最終加壓濃度為125nM。MTX加壓程過程中采用96孔板倍比稀釋的方法篩選加壓后的SIgA單克隆細(xì)胞系,獲得表達(dá)量較高的細(xì)胞株。 4分泌型IgA的發(fā)酵培養(yǎng)及純化 將SIgA單克隆細(xì)胞無血清培養(yǎng)馴化2個(gè)月,采用CD CHO培養(yǎng)基(含一定濃度的MTX),逐漸降低DMEM和透析血清濃度,直至其完全適應(yīng)CD CHO生長;懸浮培養(yǎng)2-4周,培養(yǎng)基仍為CD CHO(含加壓濃度的MTX)。收集細(xì)胞培養(yǎng)上清1L左右利用Protein-L的親和層析柱進(jìn)行純化。 5分泌片SC單克隆抗體的制備 在SIgA抗體檢測過程中,需要大量SC單抗,我們首先構(gòu)建了原核表達(dá)載體pQE80L-SC,然后進(jìn)行SC蛋白的誘導(dǎo)表達(dá),SC蛋白經(jīng)過純化回收免疫小鼠,制備了2株單克隆抗體,并利用這些抗體建立了SIgA的快速診斷檢測方法。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
本文編號：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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
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本文編號：2141913
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