甲型流感病毒H1N1 HA 蛋白在果蝇S2 细胞中的表达及免疫原性研究

目的 在果蝇S2细胞表达甲型流感H1N1 HA并分析其免疫原性。方法 根据GenBank发表的甲型流感病毒（H1N1）HA基因序列,经过密码子优化,全基因合成编码HA的基因,并于其N端引入特定的空间折叠序列,定向连接至表达载体pMT/ Bip/ V5-His A,构建重组表达载体pMT-HA。酶切鉴定正确后,用脂质体转染法与辅助质粒pCoHygro 共转染果蝇S2细胞,潮霉素加压筛选稳定细胞系,在无血清培养基中以硫酸铜溶液诱导表达,SDS-PAGE电泳及Western blot鉴定,经镍柱纯化后免疫小鼠,ELISA检测其诱导的特异性抗体水平。结果 获得了稳定表达HA的S2细胞株,目的蛋白以分泌形式表达于上清,并形成三聚体,可以被H1N1 HA 抗体识别;免疫小鼠后可诱导机体产生抗HA特异性抗体。结论 获得了纯化的三聚体形式表达的HA蛋白,并具有较好的免疫原性,具有很好的疫苗应用前景。     

猪链球菌2型甲基受体趋化蛋白编码基因05SSU0273的原核表达及纯化

目的 原核表达猪链球菌2型甲基受体趋化蛋白编码基因05SSU0273。方法 通过PCR方法检测该基因在不同血清型猪链球菌中的分布情况,然后利用获得的猪链球菌2型05SSU0273基因片段, 构建重组表达载体pET32a::05SSU0273。将质粒转入E. coli BL21中,经IPTG诱导,SDS-PAGE和Western blot实验证实目的基因的表达, 然后以His亲和层析柱纯化重组蛋白。结果 在S. suis 2 35个血清型标准株中,有20株分离株扩增出目的条带。重组质粒可在宿主菌中高效表达,经镍柱亲和层析得到相对分子质量为43 kD的重组蛋白。结论 本实验成功获得S. suis 2 MCP蛋白,为研究其在猪链球菌2型致病过程中发挥的作用奠定了基础。     

华支睾吸虫CsSCCRO基因的克隆表达和蛋白纯化

目的原核表达华支睾吸虫鳞状上皮癌相关肿瘤基因(SCCRO)全长编码区序列,获得纯化的重组蛋白,为进一步功能研究做好准备。方法用生物信息学方法从华支睾吸虫成虫全长cDNA文库中识别出与人鳞状上皮癌相关基因的同源序列及其全长编码区,将其编码区序列克隆到原核表达载体PET-30a(+),测序鉴定重组质粒,在大肠杆菌BL-21/DE3中用IPTG诱导表达,重组产物用His-镍蛋白纯化柱纯化。结果华支睾吸虫鳞状上皮癌相关基因长度为1 218bp,其全长编码序列长度为780bp,编码259个氨基酸,1-22位为分泌信号肽,在羧基端有一个高度保守的区域。该基因在大肠杆菌中得到了高效的可溶性表达,重组蛋白达总蛋白的39%,纯化后的重组蛋白纯度可达98%。结论鳞状上皮癌相关基因的PET-30a(+)原核重组质粒在大肠杆菌BL-21/DE3中可以稳定高效地表达可溶性蛋白。     

结核分枝杆菌分泌蛋白MPT64单克隆抗体的制备及其特异性

目的 制备结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌蛋白MPT64的单克隆抗体(Monoclonal antibody,mAb),并分析mAb的特异性。方法 PCR扩增mpt64基因并克隆入pET28a(+)构建原核表达载体;将获得的重组菌株在IPTG作用下诱导表达MPT64蛋白,Western blot验证蛋白表达;亲和层析法纯化目的蛋白。重组MPT64蛋白免疫小鼠,取脾细胞与杂交瘤细胞SP2/0融合,筛选得到阳性杂交瘤细胞系。以该杂交瘤细胞制备小鼠腹水,中压液相色谱仪纯化腹水获得mAb。ELISA法检测获得的mAb的相对亲和力和亚类,Western blot检测mAb的特异性。结果 本研究成功构建原核表达载体pET28a(+)-mpt64并诱导表达了MPT64蛋白。亲和层析法获得纯化的重组MPT64蛋白。该重组蛋白免疫小鼠的脾细胞和SP2/0细胞融合后,经筛选获得能够稳定分泌抗MPT64 mAb的杂交瘤细胞系MPT64-A5B2。中压液相色谱仪纯化小鼠腹水中的MPT64-A5B2 mAb。该mAb的亲和力为2.813×10^-7 g/mL,属于IgG 1亚类。MPT64-A5B2 mAb能特异性识别Mtb中的MPT64蛋白。结论 本研究制备了MPT64重组蛋白,并获得了抗MPT64 mAb,为MPT64用于结核病诊断和治疗制剂的研制奠定了基础。     

旋毛虫与人骨肉瘤细胞MG-63相关抗原基因TCTP的原核表达与鉴定

目的对旋毛虫(Trichinella spiralis)与人骨肉瘤细胞MG-63相关抗原基因TCTP进行原核表达,对表达产物进行鉴定及反应原性分析。方法利用抗MG-63细胞全蛋白抗血清筛选旋毛虫肌幼虫cDNA表达文库得到旋毛虫TCTP基因。PCR扩增TCTP基因,连接至pET-32a(+)载体,构建重组质粒pET-32a(+)-TCTP,转化入感受态细胞BL21(DE3)后用IPTG诱导表达,利用His标签镍离子蛋白纯化柱对表达产物进行纯化,采用SDS-PAGE和Western blot鉴定重组蛋白的表达及反应原性。结果经酶切鉴定和测序鉴定,重组质粒pET-32a (+)-TCTP构建正确。SDS-PAGE检测重组蛋白TCTP在原核表达系统中主要以可溶性形式表达,融合蛋白的相对分子质量约为43×10~3。Wetern blot检测重组蛋白TCTP可被抗MG-63细胞多抗血清识别。结论成功构建了重组质粒pET-32a(+)-TCTP,表达的重组蛋白TCTP具有反应原性,为该蛋白生物学功能的研究奠定了基础。     

屋尘螨重组变应原Der p 2的表达、纯化及免疫学活性鉴定

目的大量诱导表达含pET24a-Derp2质粒的BL21工程菌,表达产物以重组蛋白包涵体的形式存在,经包涵体洗涤与溶解后,使用结合6组氨酸的镍柱进行亲和层析纯化蛋白质,用梯度复性方法进行重组蛋白的复性,再用屋尘螨过敏性哮喘患者阳性血清经Western blot方法分析Derp2重组蛋白的免疫学特性。纯化后的融合蛋白Derp2具有较高的纯度及较强的免疫活性,可望作为有效的屋尘螨变应原诊断试剂和疫苗的候选分子。     

2型猪链球菌细胞分裂蛋白GpsB的原核表达及其鉴定

目的 利用原核表达系统诱导表达2型猪链球菌(Streptococcus suis 2,S.suis 2)分裂相关因子GpsB重组蛋白,为后续研究奠定基础。方法 以S. suis 2菌株05ZYH33全基因组DNA为模板,经PCR扩增得到目的基因片段。目的基因经双酶切后连接至表达载体pET32a,转化大肠杆菌(Escherichia coli, E. coli)DH5α感受态细胞。重组质粒经测序鉴定正确后转化E. coli BL21感受态细胞。获得的重组表达菌经IPTG诱导表达目的蛋白。利用Ni离子亲和层析柱纯化重组蛋白,并进行SDS-PAGE和Western blot 鉴定。利用重组蛋白免疫小鼠制备多克隆抗体。结果 成功构建出重组表达载体pET32a^gpsB,并经IPTG诱导表达出目的蛋白。重组蛋白主要存在于表达菌裂解液上清中,分子质量约30 kD,与预期大小一致。Western blot 检测发现,该蛋白能被His-Tag 单克隆抗体特异性识别。制备的多克隆抗体能特异性识别重组GpsB蛋白(rGpsB)。结论 成功表达和纯化了rGpsB并获得了该蛋白的多克隆抗体,为进一步研究该蛋白在S.suis 2 分裂过程中的作用鉴定了基础。     

Tumstatin-EGFP真核表达载体构建及稳定转染CHO细胞系的建立

目的 构建重组tumstatin-增强型绿色荧光蛋白(EGFP)的分泌型真核表达载体质粒,建立稳定转染的中国仓鼠卵巢细胞系(CHO-K1).方法 利用重叠多聚酶链反应(PCR)技术从pGEM-T/STL质粒和pEGFP-C2质粒中扩增出STL-EGFP,用DNA重组技术将片段定向插入pIRESneo3质粒中,经酶切和序列验证正确后,用lipofectamine 2000转染CHO-K1细胞,通过G418筛选建立稳定转染的CHO细胞系,用逆转录-PCR、Western blot检测tumstatin-EGFP的表达,用倒置荧光显微镜观察绿色荧光蛋白的表达.结果 重组真核表达载体正确构建并在CHO细胞获得稳定表达,tumstatin-EGFP基因整合入细胞基因组DNA,培养液上清有融合蛋白tumstatin-EGFP的分泌,绿色荧光蛋白的表达高达95%以上.结论 成功构建了质粒PIRESneo3-STL-EGFP,获得稳定表达tumstatin-EGFP的细胞系.     

重组结核杆菌热休克蛋白Acr2的原核表达、纯化及鉴定的研究

目的原核表达、纯化结核杆菌热休克蛋白Acr2分子,并分析其免疫反应性。方法采用PCR法扩增结核杆菌Acr2基因,并构建重组表达质粒PET22b-Acr2,转化大肠埃希菌BL21(DE3)菌株,并用IPTG诱导Acr2蛋白表达。以及使用镍离子亲和层析柱纯化该表达产物,并通过Western blot检测其免疫反应性。结果 PCR、双酶切和测序结果均表明PET22b-Acr2重组质粒构建成功,其在大肠埃希菌中主要以包涵体蛋白形式进行表达。复性后的包涵体蛋白经镍离子亲和层析柱纯化后,可获得纯化的Acr2重组蛋白,该重组蛋白分子质量约为18.3ku,与结核分枝杆菌Acr2蛋白的理论预测值相符。经Western blot检测显示该重组蛋白能被结核病人血清特异性识别。结论原核表达质粒PET22b-Acr2被成功构建,以及Acr2重组蛋白被有效进行表达和纯化,从而为进一步研究其生物学功能奠定了基础。     

4种血清型登革病毒多表位重组抗原的表达及血清学初步评价

目的在原核表达系统中对登革病毒包膜蛋白(E蛋白)和非结构蛋白1(NS1)融合的B细胞抗原表位进行表达、纯化及血清学评价。方法将B细胞抗原表位用形成α螺旋的连接肽(EAAAK)2作为接头,串联合成1条全新的多表位融合重组基因rE,将其克隆到原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3)后经IPTG诱导表达融合蛋白,用镍柱对重组蛋白纯化,并用SDS-PAGE和Western blot方法鉴定表达产物。以融合蛋白为抗原,用间接ELISA检测登革热病人血清IgM抗体。结果重组表达载体pET28a-rE构建成功,并在大肠杆菌BL21(DE3)中成功表达。融合蛋白主要以包涵体形式存在,用镍柱纯化获得高纯度的目的蛋白,SDS-PAGE和Western blot检测结果显示蛋白分子量大小与预期结果相符,建立的间接ELISA具有较高的准确性。结论原核表达的登革病毒多表位融合蛋白具有良好的血清学检测价值。     

Receptor-binding domain of SARS-Cov spike protein： Soluble expression in E.coli,purification and functional characterization

AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS: Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli(BL21(DE3) and Rosetta-gamiB (DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST·RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST·RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21 (DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.     

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in purification and functional characterization

AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.METHODS: Three fusion tags (glutathione S-transferase,GST; thioredoxin, Trx; maltose-binding protein, MBP),which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli( BL21( DE3 ) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2)positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and respectively.CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E. coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E. coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.     

Identification and Comparison of Receptor Binding Characteristics of the Spike Protein of Two Porcine Epidemic Diarrhea Virus Strains

Porcine epidemic diarrhea virus (PEDV), a member of Alphacoronavirus, has caused huge economic losses for the global pork industry recently. The spike (S) protein mediates PEDV entry into host cells. Herein, we investigated the interactions between the S protein and its receptor porcine aminopeptidase N (pAPN) or co-receptor sugars. The C-terminal domain (CTD) of the S1 domain is bound to pAPN. The prototype strain demonstrated similar receptor-binding activity compared with the variant field isolate. Three loops at the tips of the β-barrel domains did not play crucial roles in the PEDV S-pAPN association, indicating that PEDV conforms to a different receptor recognition model compared with transmissible gastroenteritis virus (TGEV), porcine respiratory CoV (PRCV), and human coronavirus NL63 (HCoV-NL63). The N-terminal domain (NTD) of the PEDV S1 domain could bind sugar, a possible co-receptor for PEDV. The prototype strain exhibited weaker sugar-binding activity compared with the variant field isolate. Strategies targeting the receptor binding domain (RBD) may be helpful for developing vaccines or antiviral drugs for PEDV. Understanding the differences in receptor binding between the prototype and the variant strains may provide insight into PEDV pathogenesis.     

Tale of two spikes in bacteriophage PRD1

Structural comparisons between bacteriophage PRD1 and adenovirus have revealed an evolutionary relationship that has contributed significantly to current ideas on virus phylogeny. However, the structural organization of the receptor-binding spike complex and how the different symmetry mismatches are mediated between the spike-complex proteins are not clear. We determined the architecture of the PRD1 spike complex by using electron microscopy and three-dimensional image reconstruction of a series of PRD1 mutants. We constructed an atomic model for the full-length P5 spike protein by using comparative modeling. P5 was shown to be bound directly to the penton base protein P31. P5 and the receptor-binding protein P2 form two separate spikes, interacting with each other near the capsid shell. P5, with a tumor necrosis factor-like head domain, may have been responsible for host recognition before capture of the current receptor-binding protein P2.     

Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein

Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion–integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.     

SARS病毒Spike蛋白重组腺病毒疫苗的构建及其免疫学分析

目的构建基于sARS病毒Spike（S）蛋白的重组腺病毒,并对该重组腺病毒进行初步鉴定和免疫学探讨。方法通过全基因合成得到全长S蛋白基因,利用Ad5腺病毒系统构建S蛋白基因的重组腺病毒,采用间接免疫荧光（IFA）试验和蛋白免疫印迹法（Western blot）对S蛋白的表达进行鉴定,再用重组腺病毒免疫小鼠,观察小鼠抗体诱生情况,从而对该重组腺病毒的免疫效果进行初步评价。结果PCR扩增重组腺病毒质粒外源片段,大小为800bp,与预期值一致;Western blot和IFA结果表明,重组腺病毒（rAd-S）表达S蛋白能被马抗SARS血清识别;ELISA检测结果显示,rAd-S能刺激小鼠机体产生抗体。结论重组腺病毒系统成功表达了SARS病毒S蛋白,免疫小鼠产生S蛋白特异的抗体。     

Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.

De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response. Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH). A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli [Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B. & Murphy, J.R. (1987) Protein Eng. 1, 493-498]. We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH. The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent. Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells. Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes. DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid.     

Prognostic Significance of Receptor-Binding Cancer Antigen Expressed on SiSo Cells (RCAS1) Expression in Relation to Cadherin Expression in Patients with Colorectal Carcinoma

Purpose This study was designed to assess the prognostic value of receptor-binding cancer antigen expressed on SiSo cells expression and its relationship with cadherin expression in patients with colorectal cancer. Methods The expressions of receptor-binding cancer antigen expressed on SiSo cells and E-cadherin were analyzed with special reference to prognosis in 105 patients with colorectal cancer. Results Receptor-binding cancer antigen expressed on SiSo cells immunoreactivity was detected in the membrane and cytoplasm of tumor cells and considered to be positive in 48 patients (45.7 percent). The expression of receptor-binding cancer antigen expressed on SiSo cells was significantly correlated with lymph node metastasis (P = 0.0004), venous invasion (P = 0.0062), Dukes stages (P < 0.0001), and serum levels of carcinoembryonic antigen (P = 0.014). Furthermore, receptor-binding cancer antigen expressed on SiSo cells expression was significantly correlated with a poor prognosis (P < 0.001), and multivariate analysis indicated that it was an independent prognostic indicator. The expression of receptor-binding cancer antigen expressed on SiSo cells was more frequently found in tumors with reduced or abnormal expression of E-cadherin. The survival time of patients with reduced/abnormal E-cadherin expression was significantly shorter than that of patients with normal E-cadherin expression among patients with receptor-binding cancer antigen expressed on SiSo cells expression (P = 0.0043) but did not differ for those without receptor-binding cancer antigen expressed on SiSo cells expression (P = 0.17). Furthermore, multivariate analysis revealed that reduced/abnormal expression of E-cadherin was an independent prognostic factor in patients with receptor-binding cancer antigen expressed on SiSo cells expression but not in those without receptor-binding cancer antigen expressed on SiSo cells expression. Conclusions Receptor-binding cancer antigen expressed on SiSo cells expression is significantly correlated with tumor progression and poor prognosis in patients with colorectal cancer. Both reduced E-cadherin and enhanced receptor-binding cancer antigen expressed on SiSo cells expression may be critical for the mechanism of metastasis and recurrence in human colorectal cancer.     

SARS冠状病毒S蛋白部分序列1的克隆与表达

目的　构建SARS冠状病毒S蛋白部分序列 1(S1)的原核重组表达质粒 ,并分析其在大肠杆菌中的表达状况。方法　采用逆转录 -多聚酶链反应 (RT -PCR)技术从SARS冠状病毒RNA中扩增出编码S1蛋白的基因片段 ,并克隆至pMD18-T载体上 ;菌落PCR鉴定阳性克隆并测序列分析 ;将阳性克隆的插入片段亚克隆至表达载体pGEX - 4T - 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定转化菌落 ;将阳性菌株经IPTG诱导 ,SDS -PAGE和免疫印迹分析截短型S蛋白的表达。结果　RTPCR扩增出S1蛋白的特异片段 ,其阳性克隆的序列存在 2处碱基突变第 36位G变为A ;第 333位由C变为T) ,其中第 36位为有义突变 ,第 333位为同义突变 ;阳性克隆中截短型S1蛋白的基因片段被亚克隆到表达载体pGEX - 4T - 2而构建成重组表达质粒 ,并在JM 10 9中表达了S1蛋白的融合蛋白 ,表达的蛋白能与GST免疫血清和SARS病人血清反应。结论　成功构建了SARS冠状病毒S1蛋白的重组表达质粒 ,该质粒在大肠杆菌中表达了截短型S蛋白的融合蛋白。     

From Multiplex Serology to Serolomics—A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86–100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96–100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.