Stable high-level expression of truncated human papillomavirus type 16 L1 protein in Drosophila Schneider-2 cells

To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1 protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE, and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per 1×10⁷ cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVL1 protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV16VLP vaccine.     

人乳头瘤病毒16L1-减毒志贺氏杆菌为载体疫苗的构建及免疫效应初步鉴定

为预防高危型人乳头瘤病毒16型(HPV16)诱发宫颈癌,制备以减毒志贺氏杆菌为载体的HPV16预防疫苗,以期载体可介导机体产生粘膜免疫反应,达到预防HPVl6感染的目的。为此构建了以HPV16L1为免疫原的减毒志贺氏杆菌苗,并初步鉴定候选疫苗的减毒特性和免疫效果。利用基于志贺氏杆菌virG／icsA基因的表达载体(pHS3199),将HPV16L1基因插入后构成pHS3199-hpv16L1质粒,电穿孔法将其转入减毒志贺氏杆菌sh42,经筛选获得重组减毒sh42-HPV16L1工程菌。用免疫印迹法检测HPV16L1蛋白表达,连续传代法确定其传代和目的蛋白表达的稳定性;豚鼠角膜巩膜炎症试验检测细菌的毒力和菌苗的免疫效果;小鼠红细胞凝集抑制试验检测免疫血清对病毒样颗粒(VLP)的中和活性。免疫印迹检测证实,重组菌株sh42-HPV16L1可稳定表达HPV16L1;豚鼠角膜巩膜炎症试验证实,该候选菌苗无致病性。减毒sh42-HPV16L1经结膜囊途径免疫豚鼠,可以产生特异性体液免疫应答,免疫动物体内的血清、肠道、阴道分泌物中抗HPV16L1 VLPIgG、IgA含量显著高于对照组,并且sh42-HPV16L1免疫动物血清可明显抑制HPV16L1 VLP引起的小鼠红细胞凝集。因而sh42-HPV16L1将是一种潜在的HPV16候选预防疫苗。     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

截短型人乳头瘤病毒58型L1蛋白的表达及其体外生物活性研究

利用PCR技术克隆截短型HPV58 L1基因并重组入杆状病毒表达系统穿梭质粒pFastBac-Htb,通过转座反应,将目的基因片段重组入杆状病毒基因组,分离重组的Bacmid DNA, 并转染Sf-9昆虫细胞,收集被转染的Sf-9细胞,提取细胞蛋白,SDS-PAGE检测可见在大约58Kda处出现一新生蛋白条带,Western blot 证实为HPV58L1蛋白。用ProBondTM纯化系统纯化所表达的蛋白。小鼠红细胞凝集试验证实纯化的蛋白可介导小鼠红细胞凝集,透射电镜观察证实纯化蛋白可自组装成VLP。结果表明昆虫杆状病毒表达系统可高效表达截短型HPV58L1蛋白,纯化后的截短型HPV58L1蛋白在体外可自组装VLP,并具有介导小鼠红细胞凝集的生物学活性。     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1 VLP两种抗原在检测宫颈癌抗 16L1或VLP抗体及在宫颈癌血清学诊断意义上的差别 ,应用PCR技术从宫颈癌组织的DNA中扩增出全长15 35bp的HPV16L1基因片段 ,克隆至 pUC18 T载体中 ,进行DNA测序鉴定。然后 ,将HPV16L1基因克隆至pGEX 2T表达载体中 ,并诱导表达HPV16L1融合蛋白 ,分子量为 83kD ,能被HPV16L1单克隆抗体所识别。经GST柱层析法纯化后 ,与重组腺病毒表达的HPV16L1 VLP分别经酶联免疫吸附 (ELISA)法检测 12份宫颈癌患者和 35份献血员血清。 12例宫颈癌血清标本中 ,抗HPV16L1蛋白的抗体阳性率为 7例 (占 5 8.3% ) ;抗HPV16L1 VLP的抗体阳性率为 8例 (占 6 6 .7% )。经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体IgG( )的 7份患者血清 ,利用HPV16L1 VLP试剂盒检测均阳性 ;经大肠杆菌表达的重组抗原检测为HPV16抗体IgG( )的 5份患者血清 ,利用HPV16L1 VLP试剂盒检测有 1份阳性。两者对HPV16抗体的阳性检出率并无显著差异 (P >0 .0 5 )。本实验结果说明HPV16与宫颈癌高度相关 ,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1 VLP重组抗原检测抗体的敏感性并不受影响。利用重组抗原HPV16L1对宫颈癌的抗体进行定性、定量分析有助于该疾病     

Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.     

HPV 58 L1改造基因的表达及其VLP产物抗血清制备

高危型人乳头瘤病毒（human papillomavirus, HPV）慢性持续性感染是诱发宫颈癌的主要病因．体外表达的HPV主要衣壳蛋白（L1）可自组装成病毒样颗粒（virus-like particle, VLP）,免疫后可诱导产生型别特异性中和抗体,有效保护机体免受同型病毒的感染,因此可望预防病毒感染及感染相关的宫颈癌等病变．HPV 58是诱发我国妇女宫颈癌的主要高危型病毒之一,目前尚无针对HPV 58的疫苗问世．本研究联合采用多种策略对HPV 58 L1野生型基因进行改造,获得HPV 58 L1改造基因,命名为HPV 58mL1,用杆状病毒 昆虫细胞表达系统进行HPV 58 mL1的表达,CsCl密度梯度离心法纯化获得HPV 58 mL1重组蛋白,电镜分析结果显示,重组蛋白形成直径约55 nm的VLP．皮下免疫新西兰兔和豚鼠,ELISA检测显示,免疫动物产生高滴度针对HPV 58 mL1 VLP的抗血清,免疫斑点印迹检测显示,抗血清是针对VLP表面表位的．本研究表达了均一性好的HPV 58 mL1 VLP,并获得两个种属的HPV 58 mL1 VLP抗血清,为进一步研究有效预防HPV 58感染的疫苗打下基础．     

The choice of resin-bound ligand affects the structure and immunogenicity of column-purified human papillomavirus type 16 virus-like particles

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.     

Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli

Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography.     

人乳头瘤病毒16型病毒样颗粒的制备及其免疫原性研究

利用PCR技术从HPV16阳性阴道分泌物标本中获得HPV16 L1基因片段,并将其插入表达载体pTO-T7中,构建重组表达质粒pTO-T7-HPV16-L1;以该重组质粒转化大肠杆菌ER2566并表达HPV16 L1蛋白;所表达的HPV16 L1蛋白经过硫酸铵沉淀、离子交换层析和疏水相互作用层析等纯化步骤后,HPV16 L1纯度达到98%以上,并可在体外装配为直径50nm的病毒样颗粒;动物免疫原性研究结果显示,该病毒样颗粒可诱导高滴度的针对HPV16的中和抗体。上述研究结果表明通过大肠杆菌表达系统制备的HPV16病毒样颗粒具有纯度高,与天然病毒颗粒形态高度相似的特点,并具有高度免疫原性,可以应用于HPV16病毒样颗粒的结构功能研究及HPV16疫苗研发等领域。     

原核表达系统制备HPV58L1 VLP可诱发高滴度且持久的中和抗体

目的:采用大肠杆菌表达系统制备人乳头瘤病毒58型(human papillomavirus type 58,HPV58)病毒样颗粒(virus-like particle,VLP)疫苗。方法:合成法获得HPV58 L1大肠杆菌密码子优化基因,构建HPV58 L1重组原核表达质粒mpET22b/HPV58 L1,检测其在BL21(DE3)中表达水平,饱和硫酸铵沉淀加阳离子交换层析法纯化蛋白后进行动态光散射(dynamic light scatter,DLS)分析。小鼠免疫后,检测免疫血清针对HPV58假病毒的中和抗体水平。结果:HPV58 L1蛋白在BL21(DE3)细胞中大部分以可溶形式表达,纯化获得的HPV58 L1蛋白可组装成水动力学直径约为74 nm的VLP。0.5μg的HPV58 L1 VLP可诱发小鼠产生高滴度的HPV58特异性中和抗体,可维持至少20周。结论:原核表达系统制备的HPV58 L1 VLP可诱发高滴度且持久的中和抗体,可用于成本低的HPV58疫苗的研究。     

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.     

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.     

HPV18 E7致癌蛋白的高效表达、纯化和鉴定

目的为进一步研究人乳头状瘤病毒18（Human papillomavirus18,HPV18）E7蛋白的结构与功能。方法构建HPV18 E7的谷胱甘肽S-转移酶融合蛋白质粒pGEX-6P-1-GST-HPV18 E7,重组质粒转入大肠埃希菌BL21进行可溶性融合蛋白的高效表达。结果柱上切除法去除GST标签,表达产物经glutathione Sepharose 4B亲和层析纯化,获得了SDS-PAGE和HPLC-ESI-MS纯度的HPV18 E7均质蛋白,非变性PAGE和凝胶过滤表明HPV18 E7以稳定的单体形式存在于水溶液中。高压液相色谱-电喷雾质谱（HPLC-ESI-MS）分析得到HPV18 E7精确分子量为12865．0 Da,与其理论值吻合。纯化蛋白经HPLC-ESI-MS／MS鉴定为目的产物,鉴定出的9个匹配肽段覆盖率为HPV18 E7整个氨基酸序列的96．5％。结论本文所建立的技术可以有效地大量制备HPV18 E7,为进一步研究其结构与功能和致癌机制奠定了重要的物质基础。     

Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor.

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination.     

Expression, purification, and antibody binding activity of human papillomavirus 16 L1 protein fused to maltose binding protein

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.     

One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin

The 6xHis/Ni-NTA system allows rapid and efficient affinity purification of recombinant proteins from virtually any expression system. Protocols and tips for purification under both native and denaturing conditions are provided, as well as a rapid spin procedure for protein minipreps.     

HPV16 L1蛋白在大肠杆菌中的表达、纯化及免疫原性研究

用IPTG诱导目的工程菌pQE31-HPV16L1/M15(pREP4),对表达产物进行SDSPAGE和Western blot分析;用表达的L1蛋白免疫BAL B/C小鼠得到抗血清后,利用真核源性的VLP粗提物验证小鼠抗血清的特异性.利用IMAC金属亲和层析柱纯化L1蛋白.SDSPAGE结果显示表达产物在约57 ku处有蛋白条带;Western blot结果证实此条带可与HPV16 L1蛋白的单克隆抗体反应;纯化后的L1蛋白也同样保留免疫特异性;小鼠抗血清可与HPV16L1 VLP(病毒样颗粒)发生特异性反应,证实重组表达的L1蛋白具有免疫原性.本实验表明HPV16 L1蛋白在工程菌M15(pREP4)中高效表达,为研制HPV16预防性基因工程疫苗和感染的诊断试剂提供了物质基础和技术方法.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.