病毒包膜蛋白质类

摘要： 本文综述了HBV大、中、小表面蛋白相关研究进展,包括基因结构、特点、检测方法和临床意义等方面。截至目前,关于HBV大、中、小表面蛋白的临床意义尚不明确,亟需进一步研究探讨。

摘要： 目的 建立表达寨卡病毒E蛋白或含prM蛋白的G蛋白缺失型重组水疱性口炎病毒(rVSV)载体疫苗,评价其免疫效果.方法 选取E蛋白或含prM蛋白基因,分别构建入prVSVΔG和prVSV骨架质粒,通过反向遗传学操作得到2种缺失VSV-G蛋白的重组病毒疫苗(rVSV△G-E、rVSV△G-prM-2A-E)和2种含VSV-G蛋白的重组病毒疫苗(rVSV-E、rVSV-prM-2A-E),免疫BALB/c小鼠后进行体液免疫及细胞免疫水平检测,比较4种重组病毒疫苗免疫差异.结果 rVSV△G-prM-2A-E免疫后诱导产生的特异性抗体与rVSV-prM-2A-E免疫后抗体水平相当(P>0.05),但显著高于rVSV△G-E免疫组(P0.05);rVSV△G-prM-2A-E及rVSV△G-E的扩增需要添加VSV-G.结论 提供额外的VSV-G可成功包装并扩增rVSV△G-prM-2A-E,且该疫苗诱导产生较高中和抗体及细胞免疫反应,可进一步研发成实用疫苗.

摘要： 目的：分析人/猴嵌合免疫缺陷病毒（SHIVSF162P3）包膜蛋白N糖基化位点的数量和分布及病毒传播能力。方法两只雌性成年（4周岁）中国恒河猴，编号Rh1和Rh2，体重分别为4和5 kg。通过静脉攻毒的方式向Rh1和Rh2接种SHIVSF162P3，每只剂量为300半数组织培养感染剂量，在攻毒后第3、7、10、14、17、21、24、28、35、42、49、56、63、70和77天采集血液样本。对样本进行病毒RNA抽提和cDNA合成，采用实时荧光定量PCR测定病毒RNA水平。对攻毒后第7、14、28和77天的血浆样本进行单基因组扩增，使用Bio-edit中的Clustal W软件进行包膜蛋白全长多序列比对，用MEGA6.06软件计算配对差异距离，采用HIV Databases中的软件计算包膜蛋白N糖基化位点和可变区氨基酸数目，比较序列多样性、N糖基化位点数量的差异。结果从接种的病毒中共扩增得到55条包膜蛋白全长序列，其核苷酸序列平均配对差异距离为（0.1666±0.0963）%。病毒载量在攻毒后第10天达到峰值，lg转换值分别为7.68和7.49拷贝/ml；在攻毒后第42天，病毒载量达到调定点，lg转换值分别为4.27和4.81拷贝/ml。攻毒后第7天，Rh1和Rh2血样中包含25个N糖基化位点的病毒包膜蛋白序列的比例分别达到83%（20/24）和94%（29/31），高于接种病毒中的比例[49%（27/55）]。随着感染时间的延长，Rh1和Rh2血浆中SHIVSF162P3病毒包膜序列的多样性逐渐增大，包含25个N糖基化位点的包膜蛋白序列比例逐渐降低，包含27个N糖基化位点病毒所占比例逐渐升高；在第28天Rh1体内包含27个N糖基化位点的序列比例达到29%（6/21），Rh2在第77天达到35%（13/37）。V1~V5区糖基化位点分析发现，N糖基化位点数目的变化主要来自于V4区。与包含27个糖基化位点的包膜蛋白序列相比，包含25个糖基化位点的病毒包膜蛋白序列在V4区缺失7个氨基酸（TWNNTIG），导致V4区在392位和396位缺少2个N糖基化位点。结论 V4区低糖基化的SHIVSF162P3在传播过程中占优势地位；而随着感染时间的延长，V4区高糖基化的SHIVSF162P3逐渐在猴体内获得复制优势。%Objective To investigate the number and distribution of N-linked glycosylation sites of simian/human immunodeficiency virus envelope proteins (SHIVSF162P3) and SHIV transmission. Methods Two female adult Chinese rhesus macaques (4 years old) were intravenously inoculated with 300 TCID50 SHIVSF162P3. The macaques weighed 4 and 5 kg and were identified as Rh1 and Rh2. We collected plasma samples at days 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, 56, 63, 70 and 77 post-challenge. Subsequently, we monitored plasma viral load by real-time PCR after viral RNA isolation and cDNA synthesis. We amplified the full-length envelope gene by single genome amplification (SGA) at days 7, 14, 28 and 77. BioEdit, MEGA, and the HIV Databases were used to analyze envelope sequences. Sequence diversity and N-linked glycosylation sites were compared between virus stock and plasma viruses of the two macaques. Results A total of 55 env sequences were obtained from virus stock and their average pairwise distances were (0.166 6± 0.096 3)%. Viral loads peaked at 7.68 and 7.49 log10 copies/ml at day 10 and reached the set point at day 42 (4.27 and 4.81 log10 copies/ml). The percentages of envelope sequences containing 25 potential N-linked glycosylation sites (PNGSs) were 83%(20/24) and 94%(29/31) in Rh1 and Rh2, respectively, at day 7;these were significantly higher than the proportion in SHIVSF162P3 stock (49%(27/55)). Viral diversity after infection increased with time whereas the proportion of sequences containing 25 PNGSs decreased and sequences containing 27 PNGSs gradually increased. In Rh1, the percentage of sequences containing 27 PNGSs increased to 29%at day 28 and reached 35%at day 77 in Rh2. By analyzing the number of PNGSs in the V1-V5 regions, we found that PNGS variation mainly occurred in the V4 loop. Compared with sequences containing 27 PNGSs, a seven amino acid (TWNNTIG) deletion was found in the V4 loop, which resulted in a loss of two PNGSs at positions 392 and 396. Conclusion Low glycosylation of the SHIVSF162P3 V4 loop may facilitate spread of the SHIV virus whereas viruses with highly glycosylated V4 loops showed replication advantages after infection.

摘要： Objective To establish a model for preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 (HPV 6) genome in vitro, so as to lay a foundation for studying HPV life cycle. Methods The full-length linear HPV6 genome and plasmid pEGFP-▲EGFP were electrophoretically cotransferred into hTERT cells. After selection using G418 resistance, Southern blotting was performed to determine the viral load of HPV6 in transfected cells. 3T3 J2 trophoblastic cells, type I rat-tail collagen and hTERT cells containing the full-length HPV6 genes (HPV6.hTERT cells)were mixed and cocultured on metal meshes to form skin graft-like structures. Hematoxylin and eosin (HE)staining was performed to observe the structure of formed skin grafts, an immunohistochemical assay to measure the expression of HPV6 L1 protein, and electron microscopy to observe virus particles in the skin grafts. Results The linear HPV6 gene was successfully transferred into hTERT cells, and Southern blotting showed the presence of HPV6 DNA in the transferred hTERT cells. The HPV6.hTERT cells, which were cocultured with 3T3 J2 trophoblastic cells and type I rat-tail collagen, proliferated and differentiated over time, and gradually formed skin grafts giving the appearance of verrucous hyperplasia. HE staining showed that the cocultured HPV6.hTERT cells could form typical stratified structure of skin after 7 days of cultivation, and histopathologic features of HPV infection, including obvious papillomatous hyperplasia, presence of vesicular cells, hyperkeratosis and parakeratosis, could be observed after 21 days. The immunohistochemical assay showed the expression of HPV6 L1 protein in the upper portion of skin grafts, and electron microscopy revealed the presence of HPV6 virus particles in skin grafts. Conclusions The established model for preparation of tissue-engineered skin grafts using HPV 6 genome-carrying cells provides a basis for biological studies of HPV, but its application is limited to some degree.%目的：建立人乳头瘤病毒6型（HPV6）全基因体外组织工程皮片培养模型，为进一步研究 HPV病毒周期奠定基础。方法用电转的方法，将 HPV6全长线性基因和质粒 pEGFP-▲EGFP 共转染 hTERT 细胞，G418抗性筛选，Southern 印迹法检测细胞内 HPV 病毒含量；3T3 J2滋养层细胞、I 型鼠尾胶原与含 HPV6基因的 hTERT 细胞（HPV6.hTERT 细胞）混合后，在金属网格上共同培养，逐渐形成皮片样结构。 HE 染色和免疫组化检测皮片的组织结构和 HPV6 L1蛋白表达，电镜检查皮片病毒颗粒。结果 HPV6全长线性基因成功转入 hTERT 细胞，Southern 印迹法检测细胞内含 HPV6 DNA；与3T3 J2细胞、I 型鼠尾胶原共同培养的 HPV6. hTERT 细胞随时间而增殖分化，逐渐形成具有疣状增生外观的皮片。皮片 HE 染色显示，培养7 d 即出现典型的皮肤分层结构；培养21 d 皮片可见明显乳头瘤样增生、空泡细胞、角化过度、角化不全等 HPV 感染组织病理表现。免疫组化显示皮片上部有 HPV6 L1蛋白表达。电镜检测发现皮片中存在 HPV6病毒颗粒。结论 HPV6全基因组织工程皮片培养模型为 HPV 的生物学研究提供了一个平台，但在应用上有一定的局限性。

摘要： 目的 研究埃博拉病毒包膜糖蛋白的进化和变异特征.方法 从美国生物信息中心(NCBI)数据库选取1976至2014年埃博拉病毒包膜糖蛋白氨基酸序列100条,通过多序列比对和蛋白进化树分析埃博拉病毒包膜糖蛋白的进化和变异特征.结果 1976至2014年,不同亚型埃博拉病毒包膜糖蛋白氨基酸序列间的同源性为54.00％ ～ 65.00％,同亚型包膜糖蛋白氨基酸序列同源性为95.00％ ～ 100.00％.2014年在不同地域分离到的同亚型埃博拉病毒的包膜糖蛋白氨基酸序列并不完全一致,但是变异很小,同源性达到了99.70％ ～ 100.00％.2014年来自塞拉利昂的扎伊尔-埃博拉病毒包膜糖蛋白氨基酸同源性达到了100.00％,但是米自几内亚的三条扎伊尔-埃博拉病毒糖蛋白氨基酸序列有一定变异.不同亚型埃博拉病毒包膜糖蛋白分别在进化树的不同分支上:2014年分离自塞拉利昂的扎伊尔-埃博拉病毒包膜糖蛋白在一大分支上;1976至2014年分离自塞拉利昂以外国家的扎伊尔-埃博拉病毒糖蛋白在另一个大分支上.结论 埃博拉病毒包膜糖蛋白氨基酸变异具有时间性和地域性.2014年流行于不同地域的扎伊尔-埃博拉病毒可能是同一来源的两个不同变种,没有迹象表明不同亚型埃博拉病毒之间具有“混合杂交现象”发生.%Objective To study the evolutionary and mutant characteristics of Ebora virus envelope glycoprotein.Methods A total of 100 Ebora virus envelope glycoproteins amino acid sequences isolated during 1976 and 2014 were collected from National Center for Biotechnology Information (NCBI).Multiple sequence alignment and phylogenetic tree analysis were performed to investigate the evolutionary and mutant characteristics of Ebora virus envelope glycoprotein.Results Glycoprotein amino acid sequences of Ebora virus isolated during 1976 and 2014 showed only 54.00％-65.00％ homology among different subtypes,while 95.00％-100.00％ homology in same subtypes.Ebola virus isolated from different regions in 2014 showed a 99.70％-100.00％ homology of glycoprotein amino acid sequences in the same subtype.The homology of glycoprotein amino acid sequences of Ebola-Zaire virus isolated from Sierra Leone in 2014 was 100.00％,but three strains of Ebola-Zaire virus isolated from Guinea showed diversity in glycoprotein amino acid sequences.Glycoprotein amino acid sequences of Ebola virus with different subtypes were on different branches of phylogenetic tree.Glycoprotein amino acid sequences of Ebola-Zaire virus isolated from Sierra Leone in 2014 were on one branch,and those of Ebola-Zaire virus isolated from other countries during 1976 and 2014 were on the another branch.Conclusions Glycoprotein amino acid sequences of Ebora virus vary with time and region.Ebola-Zaire virus isolated from different regions in 2014 may be two variants with the same origin,and "hybrid phenomenon" is not observed among virus of different subtypes.

摘要： Objective To explore the clinical significance of up-converting phosphor immunochromatography in detecting hepatitis B virus large envelope protein (HBV-LP) in serum of chronic hepatitis B (CHB),liver cirrhosis (LC) and primary hepatic carcinoma (PHCC) patients.Methods A new UPT-based immunochromatographic technology was employed to detect hepatitis B virus large envelope protein (HBV-LP) in serum of 260 CHB,190 LC and 45 PHCC patients; chemiluminescence was adopted to detect HBeAg; real-time fluorescent quantitative PCR was utilized to detect hepatitis B virus DNA (HBV DNA).Results The majority of CHB patients have high concentrations of HBV-LP (＞40 U/ml) ; most LC and PHCC patients have low concentrations of HBV-LP (＜ 10 U/ml) ; CHB patients,LC patients and PHCC patients are all ranked in descending order in terms of the positive rates of HBeAg,HBV DNA and HBV-LP; the positive rates of HBeAg and HBV DNA in LC and PHCC patients are lower than those in CHB patients and the difference is statistically significant (P ＜ 0.01); within the groups of LC and PHCC patients,the positive rate of HBV-LP is significantly higher than those of HBV DNA and HBeAg and the difference is statistically significant (P ＜ 0.05).Conclusion As a swift and reliable method in detecting HBV-LP levels,up-converting phosphor immunochromatography is of great value in diagnosing chronic liver disease and monitoring treatment effects.%目的 探讨上转发光免疫层析法检测的乙肝病毒外膜大蛋白(HBV-LP)在慢性乙型肝炎(CHB)、乙肝后肝硬化(LC)、乙肝原发性肝癌(PHCC)患者血清中水平变化及临床意义.方法 利用一种新的基于上转发光法的免疫层析检测技术(UPT)检测260例慢性乙型肝炎、190例乙肝肝硬化及45例乙肝原发性肝癌患者血清中乙肝病毒外膜大蛋白(HBV-LP)的含量,化学发光法检测乙肝E抗原(HBeAg)含量,实时荧光定量PCR法检测乙肝病毒核酸(HBV DNA).结果 HBV-LP阳性结果浓度水平CHB组主要以＞40 U/ml的高浓度为主,LC组和PHCC组主要集中在＜10 U/ml的低浓度范围内;HBeAg、HBV DNA和HBV-LP的阳性率均表现为CHB组＞LC组＞PHCC组,HBeAg和HBV DNA在LC和PHCC患者中的阳性率均明显低于CHB患者,差异有统计学意义(P＜0.01);HBV-LP在LC和PHCC患者中的阳性率明显高于HBV DNA和HBeAg,差异有统计学意义(P＜0.05).结论 上转发光免疫层析法检测HBV-LP水平快捷可靠并且对慢性肝病进展的诊断以及疗效监测有重要的价值.

摘要： 目的 建立定量检测患者血清HBV-大蛋白（LP）的TRFIA.方法 以抗人HBV-LP单克隆抗体和抗HBs多克隆抗体分别作为固相抗体和铕标记抗体,应用TRFIA和ELISA对标准品、66份慢性乙型肝炎患者血清、30份健康体格检查者血清中HBV-LP进行检测.分析TRFIA和ELISA的有效检测范围等,统计学比较采用χ2检验和直线相关分析.结果 标准品TRFIA检测结果示该法剂量-反应曲线呈良好线性相关（r=0.999）.ELISA法测得的正常（30份健康者血清）95％参考范围为0～1.36 mg/L.TRFIA灵敏度（最小测定值）为0.10 mg/L,30份健康血清HBV-LP的检测结果均正常,特异性100％.66份患者血清TRFIA与ELISA检测结果的r为0.800 9（P＜0.001）,两者检测的阳性率差异有统计学意义[89.4％ （59/66）与77.3％（51/66）,χ2=6.13,P＜0.01].TRFIA有效可测范围（CV＜ 10％）为1.35～2 764.00 mg/L,ELISA为10.80～691.00 mg/L.TRFIA回收率范围为95.93％ ～ 107.62％.结论 TRFIA法检测患者血清中的HBV-LP灵敏度和特异性高,检测范围宽,对早期筛选HBV患者及监测抗病毒治疗的疗效具有潜在的价值.

摘要： Objective To establish a method of TRFIA with high sensitivity and broad detecting range for serum HBV large surface protein (HBV-LP).Methods The monoclonal antibody of HBV-LP was covered on the microwell plate and incubated with HBV-LP in blood sample,then Eu3+ labeled antibody of HBs was added.HBV-LP in standard substance,blood samples of 66 chronic hepatitis B patients and 30 healthy controls was detected by the TRFIA and ELISA.x2 test and linear correlation analysis were used for data analysis.Results The dose-response curve of standard substance with TRFIA had good linear correlation (r=0.999).Normal reference range was established at 0-1.36 mg/L based on the ELISA results of 30 healthy controls.The sensitivity was 0.10 mg/L.The specificity was 100％ (30/30).Correlation coefficient between the TRFIA and the ELISA was 0.800 9 (P＜0.001).The positive detecting rates of the 2 methods were significantly different (89.4％(59/66) vs 77.3％(51/66),x2 =6.13,P＜0.01).The recovery rate for HBV-LP was between 95.93％-107.62％.The effective detecting range(CV＜10％) of TRFIA was 1.35-2 764.00 mg/L,and that of ELISA was 10.8-691.0 mg/L.Conclusion The TRFIA was established for HBV-LP detection with higher sensitivity and wider detecting range compared to ELISA.It has potential value for HBV screening and monitoring of antiviral therapy.%目的 建立定量检测患者血清HBV-大蛋白(LP)的TRFIA.方法 以抗人HBV-LP单克隆抗体和抗HBs多克隆抗体分别作为固相抗体和铕标记抗体,应用TRFIA和ELISA对标准品、66份慢性乙型肝炎患者血清、30份健康体格检查者血清中HBV-LP进行检测.分析TRFIA和ELISA的有效检测范围等,统计学比较采用x2检验和直线相关分析.结果 标准品TRFIA检测结果示该法剂量-反应曲线呈良好线性相关(r=0.999).ELISA法测得的正常(30份健康者血清)95％参考范围为0～1.36 mg/L.TRFIA灵敏度(最小测定值)为0.10 mg/L,30份健康血清HBV-LP的检测结果均正常,特异性100％.66份患者血清TRFIA与ELISA检测结果的r为0.800 9(P＜0.001),两者检测的阳性率差异有统计学意义[89.4％ (59/66)与77.3％(51/66),x2=6.13,P＜0.01].TRFIA有效可测范围(CV＜ 10％)为1.35～2 764.00 mg/L,ELISA为10.80～691.00 mg/L.TRFIA回收率范围为95.93％ ～ 107.62％.结论 TRFIA法检测患者血清中的HBV-LP灵敏度和特异性高,检测范围宽,对早期筛选HBV患者及监测抗病毒治疗的疗效具有潜在的价值.

摘要： 目的 建立人血清中乙型肝炎病毒外膜大蛋白(HBV-LP)的酶联免疫吸附试验(ELISA)检测方法.方法 辣根过氧化物酶标记HBV-LP单抗,优化各类反应液的工作浓度和各种反应条件,建立双抗体夹心的ELISA检测方法；同时评价所建立方法的灵敏度、特异性、稳定性等性能指标.结果 所建立方法的灵敏度为5 ng/ml,批内、批间变异均小于10％.在4°C和37°C条件下分别进行了3、5、7d的稳定性考察,线性相关系数均＞0.98,标准偏差＜10％.结论 成功建立了血清中HBV-LP的检测方法,该方法性能能够满足临床检测的需求.%Objective To establish enzyme-linked immunosorbent assay (ELISA) for detection of hepatitis B virus large surface protein(HBV-LP) in serum.Methods A sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of HBV-LP as the catalytic enzyme.Several reactions liquid's concentration and reaction conditions were optimized.The method was evaluated in all aspects such as sensitivity,specificity,stability and so on.Results The detection limit was 5 ng/ml.Interassay and intra-assay RSD were both less than 10％.After stored at 4°C and 37°C for 3,5,7 days,the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10％.Conclusion Established ELISA for determination of serum HBV-LP has high sensitivity and repeatability.

摘要： 本发明部分涉及针对病毒的包膜蛋白和/或可以特异性结合于病毒的包膜蛋白的氨基酸序列，以及包含一种或多种所述氨基酸序列或基本由一种或多种所述氨基酸序列组成的化合物或构建体，和具体地是蛋白和多肽。

摘要： 本发明提供了特异性结合基因型2a的HCV的包膜蛋白2、但是不与基因型1a的HCV的包膜蛋白2免疫反应的抗体。

摘要： 本发明提供一种用于检测寨卡病毒（ZV）包膜蛋白E特异性抗体的ELISA试剂盒。本发明试剂盒包含包被重组ZV包膜蛋白E的酶标板、样品稀释液、阴性对照血清、阳性对照血清、辣根过氧化物酶（HRP）标记的抗体、浓缩洗涤液、酶底物溶液和终止液。本发明试剂盒特异性高、重复性好，能够简单方便地用于寨卡病毒特异性抗体的检测，减少假阳性，能用于大规模血清学检测和流行病学调查，评估寨卡病毒的感染情况。

摘要： 本发明提供了病毒样颗粒(VLP)，其包含i)含有目的多肽(POI)和至少禽类嗜肝DNA病毒的大包膜(L)多肽的颗粒结合部分或其功能性衍生物或同源物，和ii)禽类嗜肝DNA病毒的小包膜(S)多肽或其功能性衍生物或同源物。通过将一个或多个POIs导入L多肽，所述POI将与L一起转运至主要由S多肽构成的颗粒结构中。本发明进一步提供了用于生产重组病毒样颗粒的方法。

摘要： 制成了黄病毒包膜蛋白中心单体接触界面中的突变，其调节所述黄病毒的感染性。所述突变降低包膜二聚体蛋白解离的能力。

摘要： 本发明提供了特异性结合基因型2a的HCV的包膜蛋白2、但是不与基因型1a的HCV的包膜蛋白2免疫反应的抗体。

摘要： 本发明部分涉及针对病毒的包膜蛋白和/或可以特异性结合于病毒的包膜蛋白的氨基酸序列，以及包含一种或多种所述氨基酸序列或基本由一种或多种所述氨基酸序列组成的化合物或构建体，和具体地是蛋白和多肽。

摘要： 本发明提供了一种产生在体外具有感染性的嗜肝DNA病毒毒粒的方法，此方法包括：(a)将(i)嗜肝DNA病毒基因组表达载体和(ii)包含编码泡沫病毒包膜蛋白至少一个片段的核酸的泡沫逆转录病毒包膜表达载体导入细胞；(b)培养该细胞从而产生含有泡沫病毒包膜至少一个片段的嗜肝DNA病毒毒粒，其中所述嗜肝DNA病毒毒粒在体外具有感染性。本发明还提供了测定抗嗜肝DNA病毒药物敏感性的方法，测定来源于被感染病人的嗜肝DNA病毒复制能力的方法，及鉴定嗜肝DNA病毒核酸中与抗嗜肝DNA病毒药物抗性相关的突变的方法。

摘要： 本发明提供了一种巨细胞病毒CMV包膜蛋白包装慢病毒载体及其应用，属于生物医学工程领域。本发明针对现有慢病毒载体转染NK细胞的局限性，将巨细胞病毒包膜蛋白(gH、gL和gO)转入包装细胞293T，包装慢病毒DNA产生病毒。本发明以CMV膜蛋白(gH/gL/gO)替代VSV‑G包装慢病毒假病毒，不仅维持了较高的病毒滴度，对上皮细胞具有稳定的转染功能，且能够有效识别NK细胞而且能够介导病毒进入细胞，从而实现高效转染NK细胞的目的，为基因治疗和细胞转染研究提供新的工具，可应用于大规模的研发及生产中。

摘要： 本发明提供了一种快速检测寨卡病毒(Zika virus,ZIKV)包膜蛋白E的ELISA试剂盒，并提供了一种寨卡病毒包膜E蛋白特异性抗原的筛选及特异性抗体的制备方法。该试剂盒包括捕获抗体和与辣根过氧化物酶标记物结合的检测抗体,可特异性检测寨卡病毒包膜蛋白E，与其他黄病毒属病毒,如登革病毒、西尼罗河病毒和乙型脑炎病毒无交叉反应。本发明的检测试剂盒具有快速、灵敏度高、标准化的优点，可对寨卡病毒进行早期体外诊断。