昆虫细胞偏爱密码子优化的HPV16L1基因在昆虫和哺乳动物细胞中的表达

目的获得有效表达人乳头瘤病毒16型（HPV16）L1基因的重组杆状病毒和腺病毒，为研究HPV的免疫保护机制提供材料。方法按照昆虫细胞密码子偏爱优化并合成HPV16LI基因，利用Bac—to-Bac昆虫表达系统获得表达HPV16L1基因的重组杆状病毒，利用AdEasy腺病毒载体系统获得表达HPV16L1基因的重组腺病毒载体。通过间接免疫荧光和Westernblot对HPV16L1基因表达进行鉴定，利用负染电子显微镜观察病毒样颗粒（VLP）的形成。结果获得了稳定表达HPV16L1蛋白的重组杆状病毒和重组腺病毒载体，在Sf9细胞和293细胞中可有效表达能被抗HPV16L1单克隆抗体识别的L1蛋白，分子质量单位为56ku，在Sf9细胞中可观察到VLP的形成。结论按照昆虫细胞密码子偏爱进行优化的HPV16L1基因，在昆虫细胞和哺乳动物细胞内均可有效表达。     

Cellular immune responses to human papillomavirus (HPV)-16 L1 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles

The causal association between papillomavirus (HPV) infection and cervical cancer has been demonstrated; the development of a prophylactic vaccine to protect against HPV infection may therefore reduce the incidence of this cancer worldwide. Noninfectious HPV-like particles (VLPs), composed of the L1 major capsid protein, are current candidate vaccines for prevention of HPV infection and cervical neoplasia. Although neutralizing antibodies have a pivotal role in the prevention of initial infection, cellular immune responses to HPV antigens may have an important role in viral clearance. A phase II trial was conducted to further evaluate the immunogenicity of a recombinant HPV-16 L1 VLP vaccine administered intramuscularly, without adjuvant, at 0, 1, and 6 months. Cell-mediated immune responses (lymphoproliferation and cytokine production) to HPV-16 L1 VLPs were evaluated in peripheral blood mononuclear cells (PBMCs) from 43 individuals receiving the L1 VLP vaccine and from 10 individuals receiving placebo. Vaccination resulted, at months 2 and 7 (i.e., 1 month after the second immunization and 1 month after third immunization, respectively), in increases in T cell-proliferative response to HPV-16 L1 VLPs (P<.001). In addition, significant increases in cytokine (interferon-gamma, interleukin [IL]-5 and IL-10) responses to L1 VLPs were observed after vaccination (P<.001). The strongest cytokine responses at month 7 were observed in individuals with high antibody titers at month 2, suggesting that neutralizing antibodies generated by initial vaccination may augment T cell responses to subsequent booster vaccinations. No significant increases in lymphoproliferative or cytokine responses to L1 VLPs were observed in individuals receiving placebo. In summary, the HPV-16 L1 vaccine induces not only robust B cell responses but also L1-specific T cell responses detectable by proliferation of both CD4+ and CD8+ T cells and in vitro production of both Th1- and Th2-type cytokines. Future efficacy studies are needed to evaluate whether and/or how VLP vaccines confer protection against genital HPV infection and associated disease.     

Use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization

Recombinant virus-vectored TB vaccines represent the most promising vaccine platform for boosting the protective immunity mediated by parenteral BCG prime immunization. A major advantage associated with virus-vectored vaccines is that they are potent respiratory mucosa-deliverable vaccines. A recombinant replication-deficient adenoviral (Ad) vector was engineered to express Mycobacterium tuberculosis (M.tb) Ag85A. Single administration of this Ad vaccine via the intranasal, but not intramuscular, route provided potent immune protection from pulmonary M.tb challenge. Respiratory mucosal boosting immunization with Ad vaccine was effective in enhancing T-cell activation and immune protection following parenteral DNA or BCG prime immunization. We have also recently developed a recombinant vesicular stomatitis virus-vectored (VSV) TB vaccine. Ad and VSV vector systems will be complementary to each other for BCG prime-virus vaccine boost immunization protocols.     

Vaccines against human papillomavirus and perspectives for the prevention and control of cervical cancer

Today, "persistent" infections by certain types of human papillomavirus (HPV) are considered necessary for developing cervical cancer. Producing efficient vaccines against these viruses may eventually lead to a great reduction in incidence and mortality rates of this cancer. In the case of HPV, the production of traditional vaccines usually based in dead or attenuated viruses is not possible due in part to the lack of systems where large quantities of viral particles could be obtained. Fortunately, the expression of the late L1 protein alone, or in combination with L2, leads to the generation of structures resembling true virions that have been called virus-like particles (VLPs) and constitute excellent candidates as prophylactic vaccines. VLPs have shown to be very immunogenic, and have prevented development of natural or challenged infections in both animal systems and humans. Recently, HPV16VLPs were shown to be very efficient to prevent the development of "persistent" infections, as determined by PCR assays, in a large group of vaccinated women. Therapeutic vaccines, on the other hand, are expected to have an impact on advanced lesions and residual illness, by taking advantage of the fact that early E6 and E7 genes are thought to be constitutively expressed in cervical tumors and precursor lesions. Finally, DNA-based vaccines could represent a useful alternative for preventing infections by genital HPV. This paper is available too at: http://www.insp.mx/salud/index.html.     

A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers

Viruslike particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)--11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV--11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen--specific interferon--gamma and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types.     

Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus.

As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems world-wide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitis B infection. Although considerable progress has been made in the construction of recombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and efficacy testing of candidate vaccines has remained difficult due to the lack of a suitable animal model. We demonstrate here that chimpanzees are susceptible to enteric infection by human adenoviruses type 7 (Ad7) and type 4 (Ad4) following oral administration of live virus. Moreover, after sequential oral immunization with Ad7- and Ad4-vectored vaccines containing the hepatitis B surface antigen (HBsAg) gene, significant antibody responses to HBsAg (anti-HBs) were induced in two chimpanzees. After challenge with heterologous HBV, one chimpanzee was protected from acute hepatitis and the other chimpanzee experienced modified HBV-induced disease. These data demonstrate the feasibility of using orally administered recombinant adenoviruses as a general approach to vaccination.     

HPV16 L1在整合型重组毕赤酵母中的表达及病毒样颗粒的纯化

目的 构建可稳定表达HPV16 L1的整合型重组毕赤酵母,并纯化自主组装成的HPV16 L1病毒样颗粒（VLPs）.方法 根据酵母密码子偏爱性优化HPV16 L1基因并克隆到pPIC3.5K表达载体,构建pPIC3.5K/HPV16 L1重组质粒;重组质粒经Bgl Ⅱ酶切线性化后,电转化至GS115菌株中,筛选HPV16 L1重组毕赤酵母.阳性整合菌株甲醇诱导后,以HPV16 L1单克隆抗体检测目的蛋白表达;采用肝素亲和层析法纯化HPV16 L1VLPs并进行透射电镜观察.结果 PCR、酶切和测序分析表明成功构建了pPIC3.5K/HPV16 L1重组质粒.成功构建的HPV16 L1重组毕赤酵母甲醇诱导后,Western blot证实重组酵母菌裂解产物存在HPV16 L1目的蛋白.肝素亲和纯化后,透射电镜观察到了直径大约55 nm的VLPs,其形态与HPV16天然病毒颗粒相似.结论 利用整合型重组毕赤酵母表达系统成功表达了HPV16L1蛋白,并用肝素亲和纯化可快速获得结构完整的HPV16 L1VLPs,为HPV16预防性疫苗的研制奠定基础.     

Adenovirus Biology,Recombinant Adenovirus,and Adenovirus Usage in Gene Therapy

Gene therapy is currently in the public spotlight. Several gene therapy products, including oncolytic virus (OV), which predominantly replicates in and kills cancer cells, and COVID-19 vaccines have recently been commercialized. Recombinant adenoviruses, including replication-defective adenoviral vector and conditionally replicating adenovirus (CRA; oncolytic adenovirus), have been extensively studied and used in clinical trials for cancer and vaccines. Here, we review the biology of wild-type adenoviruses, the methodological principle for constructing recombinant adenoviruses, therapeutic applications of recombinant adenoviruses, and new technologies in pluripotent stem cell (PSC)-based regenerative medicine. Moreover, this article describes the technology platform for efficient construction of diverse “CRAs that can specifically target tumors with multiple factors” (m-CRAs). This technology allows for modification of four parts in the adenoviral E1 region and the subsequent insertion of a therapeutic gene and promoter to enhance cancer-specific viral replication (i.e., safety) as well as therapeutic effects. The screening study using the m-CRA technology successfully identified survivin-responsive m-CRA (Surv.m-CRA) as among the best m-CRAs, and clinical trials of Surv.m-CRA are underway for patients with cancer. This article also describes new recombinant adenovirus-based technologies for solving issues in PSC-based regenerative medicine.     

人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。     

Adenovirus-human immunodeficiency virus (HIV) envelope recombinant vaccines elicit high-titered HIV-neutralizing antibodies in the dog model.

Recombinant human adenoviruses (Ads) (types 4, 5, and 7) expressing the HIV-1 envelope membrane glycoprotein (gp160) were tested for immunogenicity in the dog. Administration of recombinant Ad7-env by intratracheal inoculation resulted in a low serum antibody response to gp160, which developed over several weeks. A strong neutralizing antibody response to the Ad7 vector developed within 1 week of infection. A subsequent booster inoculation 12 weeks later with the heterotypic Ad4-env recombinant virus resulted in significantly enhanced humoral responses directed at the envelope antigen, as measured by both ELISA and Western blot analysis as well as high-titer type-specific neutralizing antibodies, with some animals achieving neutralization titers approaching 1000. Recombinant HIV envelope glycoprotein derived from Ad-HIV-infected cell cultures was used as a subunit booster injection for dogs that had previously received sequential immunizations with heterotypic recombinant Ads. Significant immune responses against the envelope developed as measured by ELISA, Western blot analysis, and neutralization assays. These data indicate that live recombinant Ad-HIV vaccines are capable of inducing high-titer type-specific neutralizing antibodies to gp160 in vivo. Recombinant HIV envelope glycoprotein subunit vaccines, prepared from Ad-env-infected cells, are capable of boosting these responses.     

Mixed Bacteriophage MS2-L2 VLPs Elicit Long-Lasting Protective Antibodies against HPV Pseudovirus 51

Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are highly immunogenic. The most recent HPV vaccine, Gardasil-9, protects against HPV types associated with ~90% of cervical cancer (worldwide). Thus, ~10% of HPV-associated cancers are not protected by Gardasil-9. Although this is not a large percentage overall, the HPV types associated with 10% of cervical cancer not protected by the current vaccine are significantly important, especially in HIV/AIDS patients who are infected with multiple HPV types. To broaden the spectrum of protection against HPV infections, we developed mixed MS2-L2 VLPs (MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs) in a previous study. Immunization with the VLPs neutralized/protected mice against infection with eleven high-risk HPV types associated with ~95% of cervical cancer and against one low-risk HPV type associated with ~36% of genital warts & up to 32% of recurrent respiratory papillomatosis. Here, we report that the mixed MS2-L2 VLPs can protect mice from three additional HPV types: HPV51, which is associated with ~0.8% of cervical cancer; HPV6, which is associated with up to 60% of genital warts; HPV5, which is associated with skin cancers in patients with epidermodysplasia verruciformis (EV). Overall, mixed MS2-L2 VLPs can protect against twelve HPV types associated with ~95.8% of cervical cancers and against two HPV types associated with ~90% of genital warts and >90% recurrent respiratory papillomatosis. Additionally, the VLPs protect against one of two HPV types associated with ~90% of HPV-associated skin cancers in patients with EV. More importantly, we observed that mixed MS2-L2 VLPs elicit protective antibodies that last over 9 months. Furthermore, a spray-freeze-dried formulation of the VLPs is stable, immunogenic, and protective at room temperature and 37 °C.     

A virus-based vaccine may prevent cervical cancer

High-risk human papillomaviruses (HPVs) are now recognized as the etiologic agents of invasive cervical cancer, a major cancer in women. A single HPV type (type 16) is responsible for about 50% of the cancers. The major capsid protein of papillomaviruses, L1, when expressed by recombinant DNA technology, has the intrinsic ability to assemble into virus-like particles (VLPs). In a recent study, a vaccine based on HPV 16 VLPs was tested in a placebo-controlled proof-of-principle trial in young women in the United States. The vaccine was found to prevent 100% of incident persistent HPV 16 infections and HPV 16-associated cervical intraepithelial neoplasia. These results offer promise that cervical cancer will be preventable by an HPV-based vaccine. Studies planned or in progress are examining the efficacy of the vaccine in men, in HIV-infected individuals, and in other parts of the world. Attempts are being made to prepare vaccines that can be administered more easily to large populations.     

Risk factors for subsequent cervicovaginal human papillomavirus (HPV) infection and the protective role of antibodies to HPV-16 virus-like particles

A high incidence of initial infection with human papillomavirus (HPV) was previously reported in a cohort of 608 women monitored at 6-month intervals for 3 years. Risk factors for subsequent infections with different HPV types and whether antibodies against HPV-16 virus-like particles (VLPs) protected against these infections were examined. Subsequent infections with HPV are very common. Seventy percent of women acquired a different HPV type within 24 months of the initial infection. Risk factors included being nonwhite, having an increased number of male sex partners, and having had a new male sex partner. Use of oral contraceptive pills was protective. A sustained high level of IgG antibody to HPV-16 VLPs was associated with reduced risk for subsequent infection with HPV-16 and its genetically related types (i.e., HPV-31, -33, -35, -52, and -58).     

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.     

Short communication: enhancement of immunogenicity of replication-defective adenovirus-based human immunodeficiency virus vaccines in rhesus monkeys

Adenovirus (Ad) is still under extensive investigation as a vector for HIV vaccination; however, one possible explanation for the failure of Merck's STEP trial is the relatively weak immunogenicity of replication-defective Ad vectors. In this study, a novel strategy to enhance the immunogenicity of replication-defective Ad-based HIV vaccines was developed. First, a recombinant plasmid expressing adenoviral E1 protein (pVAX-E1) was constructed to complement the E1-deleted replication-defective Ad vectors in trans. Then, the immunogenicity of the vaccine regimen of Ad5-HIV gag plus pVAX-E1 plasmid was assessed in rhesus macaques. Compared with traditional administration of Ad-based vectors alone, the results showed that our strategy elicited a more sustained and robust HIV gag-specific cellular response and enhanced long-term proliferation of CD4(+) and CD8(+) T lymphocytes. This strategy represents a proof-of-concept that enhances the immunogenicity of replication-defective Ad-based vectors, and it exemplifies the useful implications for Ad-based HIV vaccines and other vaccines.     

Evidence of prevalent genital-type human papillomavirus infections in adults and children

Recombinant proteins encoded by the E2, E7, L1, and L2 open reading frames (ORF) of human papillomavirus (HPV) types 6b, 16, and 18 were used in Western blot assays to detect serum IgG antibodies in women attending a sexually transmitted diseases clinic (n = 92) and in hospitalized children (n = 81). Antibodies to late gene products (L1 or L2 ORF) were more common than antibodies to early gene products (E2 or E7), both in the adults and the children; overall, the antibody prevalences in the children and the sexually active adults were not significantly different. Human sera with high titers of antibodies to the HPV16 E7 recombinant protein immunoprecipitated the genuine HPV16 E7 protein from the cervical carcinoma cell line CaSki. As an independent measure of HPV infection, the polymerase chain reaction was used to detect HPV6b and HPV16 in oral mucosal scrapings from adults (n = 35) and preschool children (n = 21). In adults, HPV6b and HPV16 DNA were detected in 17% and 23% of oral mucosal samples, respectively. In preschool children, HPV6b and HPV16 DNA were found in 24% and 19% of oral samples, respectively.     

CEA启动子驱动下表达Hsp70基因的重组腺病毒的构建及鉴定

目的 构建癌胚抗原(CEA)启动子驱动下靶向表达热休克蛋白70 (Hsp70)基因的重组腺病毒。方法 通过RT-PCR和PCR的方法分别扩增人Hsp70基因和CEA启动子,构建穿梭质粒pDC316-pCEA-Hsp70。将所得的穿梭质粒与腺病毒骨架质粒共转染293细胞获得重组腺病毒Ad5-pCEA-Hsp70。通过梯度离心纯化病毒,采用半数细胞培养物感染量测定病毒滴度。分别以重组的腺病毒感染CEA阳性的人胰腺癌BxPC3细胞和CEA阴性的SW1990细胞,通过RT-PCR和ELISA法检测Hsp70 mRNA和蛋白的表达。结果 酶切和测序证实Hsp70基因和CEA启动子成功插入PDC316质粒。与骨架质粒共转染293细胞获得的病毒经PCR扩增证实重组腺病毒构建成功。最终获得的病毒颗粒数达2.2 ×1011 vp/ml,滴度为1.5×1010 PFU/ml。重组腺病毒感染CEA阳性表达的BxPC3细胞后,可显著增加Hsp70 mRNA和蛋白的表达,而感染CEA阴性的SW1990细胞,Hsp70 mRNA和蛋白的表达无变化。结论 成功构建了能够在CEA阳性细胞中靶向表达Hsp70基因的重组腺病毒Ad5-pCEA-Hsp70,为进一步研究奠定了基础。     

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.     

人乳头瘤病毒16型表位嵌合病毒样颗粒的构建及免疫原性研究

目的 制备E7_49-57表位嵌合病毒样颗粒,并进行免疫原性分析。方法 利用分子模拟软件Discovery Studio预测HPV16 L1的E7_49-57最佳嵌合位点,将E7_49-57表位嵌入预测位点。构建pET28a-16L1-E7_49-57重组质粒,于大肠杆菌中诱导表达HPV16L1-E7_49-57蛋白并利用镍柱进行亲和层析纯化。于体外重组VLPs后,进行动态光散射粒径和透射电镜分析。用E7_49-57嵌合VLPs免疫小鼠,假病毒中和试验检测免疫血清中和抗体滴度。流式多因子法检测Th1和Th2型细胞因子水平。结果 HI loop区355/356为最佳表位嵌合位点。正确表达了HPV16 L1-E7_49-57蛋白并组装E7_49-57嵌合VLPs。E7_49-57嵌合VLPs免疫小鼠3次后,小鼠血清中和抗体滴度log10平均值达到4.23,略低于野生型VLPs(log10平均值为4.45)。但是,相对于野生型VLPs,E7_49-57嵌合VLPs诱导产生Th1型细胞因子(INF-γ, IL-2, TNF-α)的水平显著提高。结论 本研究制备的E7_49-57嵌合VLPs 能刺激机体同时产生较强的体液和细胞免疫应答,可能兼具预防和治疗双重功效,为宫颈癌治疗性疫苗的研制奠定基础。     

HPV 6b结构蛋白L1和沙眼衣原体CTL表位嵌合病毒样颗粒在昆虫细胞中的表达

目的为研制能同时防治人乳头瘤病毒(HPV)和沙眼衣原体(Ct)的嵌合疫苗。方法将Ct主要外膜蛋白(MOMP)插入到HPV衣壳蛋白L1羧基端,通过PCR、酶切、连接等方法构建了杆状病毒表达载体,进一步在大肠杆菌中组装成重组杆状病毒,经感染昆虫细胞表达嵌合病毒样颗粒,并经SDS-PAGE电泳和Westernblot分析鉴定,电镜负染。结果嵌合蛋白HPV6bL1/CtMOMP249-268在昆虫细胞中得到了表达,表达产物的相对分子质量为56000,与HPV6bL1单抗产生特异性反应,电子显微镜观察到病毒样颗粒形成。结论在昆虫细胞中表达的含HPV6bL1和CtCTL表位的嵌合蛋白,能自行组装成病毒样颗粒,为人乳头瘤病毒和沙眼衣原体嵌合疫苗的研究奠定基础。