• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    β-1,2-Mannan-based glycoconjugates as potential antifungal vaccines

    2022-09-15 03:11:14JunLioBoPnXioinZhuoGuochoLioYunGoZhenzhenYoLinghuWngQiuyeWuWeihuPnBinghuJioQingjieZho
    Chinese Chemical Letters 2022年9期

    Jun Lio, Bo Pn, Xioin Zhuo, Guocho Lio, Yun Go, Zhenzhen Yo,Linghu Wng, Qiuye Wu, Weihu Pn, Binghu Jio,?, Qingjie Zho,?

    a Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China

    b School of Pharmacy, Second Military Medical University, Shanghai 200433, China

    c Department of Dermatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China

    d International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

    ABSTRACT Phosphorylated di-, tri- and tetra-saccharides of β-1,2-mannan antigen derived from Candida albicans (C.albicans) cell wall were synthesized and covalently conjugated with keyhole limpet hemocyanin (KLH)and human serum albumin (HSA) via a bifunctional linker under mild conditions.The semi-synthetic β-1,2-mannoside–KLH conjugates were evaluated for the immunization of BALB/c mice.The ELISA results revealed that all three conjugates could elicit high levels of specific IgG antibodies and the acquired antisera could effectively identify the β-1,2-mannan epitope.Furthermore, the immunofluorescence and flow cytometry assays also uncovered that the induced antibodies, especially that obtained from immunization with β-1,2-mannotriose–KLH conjugate (1b), could bind well to fungi cell.Eventually, the structure–immunogenicity relationship analysis of β-mannan showed that the length of oligo-β-mannoses had a big impact on their immunogenicity and β-1,2-mannotriose showed the strongest immunogenicity.The results suggested the great potential of β-1,2-mannotriose–KLH conjugate as an antifungal vaccine candidate.

    Keywords:β-1,2-Mannan Oligosaccharide Antifungal Glycoconjugate Vaccine Antigen

    Candida albicans(C.albicans), a yeast and a common opportunistic pathogen, is one of the major causes of rising morbidity and mortality of fungal infections especially among individuals with organ transplant, cancer, and human immunodeficiency virus (HIV) infections, as well as those undergoing immunosuppressive therapy or prolonged treatment with antibiotics [1–3].In clinic, azoles, polyenes, and echinocandins are three major classes of anti-Candidadrugs; especially, triazoles are often used as firstline antifungal agents [4–7].However, with continuous abuse, fluconazole, a representative triazole, has become ineffective against invasive aspergillosis and is suffering from severe drug resistance[8–10].Thus, it is of great significance to find new strategies or methods for the treatment ofC.albicansinfection.Compared to traditional chemotherapies, vaccination is more cost-effective.For example, many vaccines against bacteria have significantly reduced the morbidity and mortality of related infectious diseases [11–14].

    For the design of anti-Candidavaccines, theβ-1,2-mannan epitope representing the carbohydrate part ofCandidacell wall phosphomannan–protein complex (PMC) [15–18] is an attractive antigen.In general, two forms ofβ-1,2-mannan can be found inCandidaspecies, which include the acid-labileβ-1,2-mannan oligosaccharides, known as antigenic factor 5, attached toα-(1,2)-mannan sidechainsviaa phosphodiester linkage and the acidstableβ-1,2-mannan, known as antigenic factor 6, as sidechains attached toα-(1,6)-mannan backbone (Fig.1) [19–24].The acidlabile phosphorylatedβ-1,2-oligomannosides are a common structure inC.albicansserotype A and B strains, whereas acid-stableβ-1,2-oligomannosides is unique to serotype A strains [25–29].These oligosaccharides are exposed on the fungal cells and function as an adhesin in the recognition of mammalian cells [19].Besides,they are highly conserved on the cell surface of pathogenic fungi[30] and thus are excellent target antigens for the development of potentially broadly useful antifungal vaccines.In the past decades,vaccines candidates that combined factor 5 or 6 or their mimics with immunogenic carrier proteins [tetanus toxoid (TT) or bovine serum albumin (BSA)], T cell peptides found inC.albicanscell wall proteins or other immunologic stimulants were synthesized, and their immunological studies indicated that both epitopes can act as antigens to induce effective and specific humoral immune responses and produce protective antibodies [31–35].Despite these great progresses, the correlations between the structures of antigenic factors, particularlyβ-1,2-mannan, and their immunogenicity are ambiguous.

    Fig.1.Two structural forms of β-1,2-mannan on the cell wall of Candida species.

    In the present work, a series of phosphorylatedβ-1,2-mannan were synthesized and covalently coupled with KLH to form glycoconjugates 1a–1c (Fig.2) as antifungal vaccine candidates.The phosphorylatedβ-1,2-mannan oligosaccharides were mimics of antigenic factor 5.Furthermore, the selectedβ-1,2-mannobiose,triose and tetraose were used to systematically investigate the structure–antigenicity relationship of linearβ-1,2-mannan.The bifunctional linker of disuccinimidyl glutarate (DSG) was used not only for efficient coupling but also as the “extended arm” anchored to the carrier protein.In the meantime, this linker was proved to not induce linker-specific antibodies to interfere with the immunological properties of the resultant conjugates.The KLH, which is derived from a circulating glycoprotein of the marine molluskMegathura crenulata, is an effective and highly immunogenic stimulant that is usually used as hapten-carrier for glycoconjugate vaccine development.In addition, the oligosaccharide–HSA conjugates 2a–2c (Fig.2) were also synthesized and used as capture reagents for detectingβ-1,2-mannan-specific antibodies.

    Synthesis of phosphorylatedβ-1,2-oligomannosides (3a–3c,Scheme 1) comprising two to fourβ-1,2-linked mannose residues are crucial for assembling the designed glycoconjugate vaccines.Herein, the methodology based on initialβ-glucosylation followed by inversion of configuration at C-2 position in the glucose residue[36–38], rather than direct mannosylation engaging conformationally rigid mannosyl donors of thioglycosides or glycosyl sulfoxides [39–41], was chosen for the consideration of highly efficient and large-scale preparation.The synthesis route was depicted in Scheme 1 and the building blocks 3a–3c were prepared according to our previous study [42].Withβ-1,2-oligomannosides 3a–3c in hand, the global deprotected compounds 4a–4c were then achieved by hydrogenolysis in the presence of Pd(OH)2/C (10 wt%)with excellent yields ranging from 88.7% to 91.5%.Thereafter, reaction of 4a–4c with a large excess of disuccinimidal glutarate 5 afforded the corresponding activated esters 6a–6c, separately, which were readily purified by filtration, precipitation with large amountof EtOAc, and washing of the precipitate several times with EtOAc in sequence.Subsequently, 6a–6c were coupled with KLH or HSA in a slightly alkaline atmosphere using phosphate buffer solution(PBS, 0.1 mol/L) to eventually provide desired glycoconjugates 1a–1c or 2a–2c, respectively, which were purified by passing through a Biogel A 0.5 column to remove remaining free sugars, dialyzing against distilled water to get rid of salts, and lyophilization.The mannose content of the glycoconjugates were analyzed by a well-known protocol of phenol-sulfuric acid method [43].The mannan-loading of KLH and HSA conjugates were displayed in Table 1, implying that the coupling reactions were efficient and in average about fifteen molecules of mannosyl were bound to each molecule of HSA.In addition, the results of mannose loadings of HSA-conjugates were also confirmed by MALDI-TOF mass spectrometry (see Supporting information).

    Scheme 1.Synthesis of glycoconjugates 1a–1c and 2a–2c.

    The immunological properties ofβ-1,2-mannan–KLH conjugates 1a–1c were evaluated in six female BALB/c mice per group.Each conjugate was combined with complete Freund’s adjuvant (CFA) as initial emulsions and then injected subcutaneously (s.c.) into mice.The next three injections of the corresponding conjugate mixed with incomplete Freund’s adjuvant were used as boost immunizations with an interval of 14 days (Fig.S1 in Supporting information).Sterile PBS and KLH were vaccinated as negative control groups, separately.One week after the final immunization, the sera from the immunized mice were collected and analyzed by enzymelinked immunosorbent assays (ELISA) for detection of the induced antibodies [44].The microtiter plates were coated with the corresponding HSA conjugates 2a–2c as capture reagents to detect theβ-1,2-mannan-specific antibodies that aimed to avoid the interference of anti-KLH antibodies.The plates were blocked with blocking buffer (5% non-fat milk in Tris-buffered saline with 0.05% Tween-20) for 2 h at 37 °C.After washing with phosphate buffered saline Tween-20 (PBST), the plates were incubated with serially diluted antisera and placed in a 37 °C incubator for 1 h.Color development of each well was achieved by secondary antibody (goat antimouse IgG-HRP, Invitrogen).The optical density (OD) at 450 and 630 nm were read using a Universal Microplate Reader (Bio-Tek Instruments, Inc.).The antibody titers were defined as the dilution number yielding an observed OD value of 0.2, and the results were shown in Fig.3.

    Fig.3.Specific IgG antibody responses of individual mouse immunized with conjugates 1a (A), 1b (B), and 1c (C), respectively.The antisera from immunized mouse with each conjugate were analyzed by ELISA with HSA conjugates 2a, 2b, and 2c as capture antigens and HRP-conjugate anti-mouse IgG antibody as the secondary antibody.#P < 0.05 vs. 1a–1c no adjuvant (Adj) group.?P < 0.05 vs. 4a–4c with adjuvant group.

    Obviously, all of the KLH-conjugates 1a–1c immunized with Freund’s adjuvant provoked higher titers ofβ-1,2-mannan-specific total IgG antibodies than that of the corresponding conjugate injected alone.It was noted that conjugates 1a–1c could elicit significant immune responses and produce high levels and antigenspecific IgG antibodies even in defect of external adjuvant, indicating that these conjugates exhibited self-adjuvanting property and may furnish the possibility of acquiring adjuvant-free vaccination.The immune response induced by 1b (average antibody titers>100,000) was notably stronger than that of 1a and 1c,which suggested thatβ-1,2-mannotriose antigen was of greatest immunogenicity.On the other hand, only very weak immune responses were elicited by freeβ-1,2-mannan oligosaccharides 4a–4c even when being combined with the Freund’s adjuvant.The similar results were also observed when administrated with the emulsion of KLH and Freund’s adjuvant.Therefore, the immunological resultants above uncovered that both the KLH and adjuvant could strengthen the immunogenicity of correspondingβ-1,2-mannan antigens to induce robust and effective immune responses.

    To further explore whether the antisera induced by 1a–1c could recognize the naturalβ-1,2-mannan antigen epitopes on the surface ofC.albicanscell, the binding affinity experiments were carried out by immunofluorescence (IF) and flow cytometry (FC).The heat-killedC.albicanscells were firstly pretreated with 3%BSA blocking buffer to exclude latently nonspecific protein binding sites on the cell surface and subsequently incubated with antisera of 1a–1c, separately.Eventually, cells were covered in Fluor 488-labeled goat anti-mouse IgG antibodies and inspected with IF and FC.As shown in Fig.4, the resultants of IF assays disclosed that the obtained antiserum from mice immunized with 1b exhibited the best binding affinity and evenly attached to cell surface of both hyphal and germtube phenotype of theC.albicansSC5314 strain whereas the negative results, hardly combining with the fungi cells, were observed for immunized antisera obtained from 1a and 1c, as well as the control group.Furthermore, we also tested the binding ability of immunized antiserum from 1b to three clinical isolates ofC.albicans(Fig.S2 in Supporting information), which were obtained from Shanghai Key Laboratory of Molecular Medical Mycology (Shanghai, China) and identified by internal transcribed spacer sequencing.The results showed that the antiserum could bind to all tested isolates, indicating that the recognition ofC.albicansis not strain-specific.

    Fig.4.Indirect immunofluorescence analysis confirmed that IgG antibodies in antisera from mice immunized with 1b could bind to the cell surface of different forms of C.albicans SC5314 strain.Antisera from mice immunized with 1a, 1c, 4a–4c, and KLH control group could not react with C.albicans SC5314 strain (Figs.S3 and S4 in Supporting information).Scale bar: 50 μm.

    The IF determinations were illustrated in Fig.5.The collected antisera from mice immunized with 1b combined with or without adjuvant exhibited satisfactory binding ability toC.albicanscell.However, the IF results of 1a and 1c were completely different from 1b.Negatively, the antiserum from mice immunized with 1c demonstrated slightly reactivity to fungus cell while the immune antisera of 1a or control group showed no binding activity toC.albicans.The IF consequences were mainly in accord with the resultants of FC suggested that the antibodies originated from immunization ofβ-1,2-mannotriose conjugate showed prominently higher reactivity toC.albicanscells than that fromβ-1,2-mannodiose conjugate, whereas the binding affinity of the induced antisera byβ-1,2-mannotetraose conjugate decreased sharply.Therefore, we may draw a simple conclusion that the length of oligo-β-mannoses plays an important role in immunogenicity andβ-1,2-mannotriose has the strongest and most effective immunogenicity.The elicited antibodies byβ-1,2-mannotriose–KLH conjugate 1b could recognize not only the syntheticβ-1,2-mannotriose oligosaccharide but also the naturalβ-1,2-mannan antigen epitope on theC.albicanscell, indicating thatβ-1,2-mannotriose rather thanβ-1,2-mannodiose or tetraose represented the unique structural motif for antigenic factor 5.We imaged that larger oligomers could provide more epitopes to induce antibodies that response toC.albicans.However, it is not a simple linear relationship between the length of manno-oligomers and immunogenicity.Only proper length of oligomers which has a reasonable spatial structure could be sufficient to induce antibodies that can recognizeC.albicans.

    In addition, the previous studies [45,46] showed that the mannans onC.tropicaliscell surface shared structural homology toC.albicansand theβ-1,2-linked oligomannosyl chains attached to the phosphate group served as the major common epitope throughoutC.albicansandC.tropicalis.Hence, it would be worthwhile to investigate the binding mode of IgG antibodies in antiserum toC.tropicalis.The IF results reflected that the antisera from mice immunized with 1b contained the antibodies that could react withC.tropicaliswhereas antibodies in antisera from mice immunized with 1a, 1c, and 4a–4c could not bind toC.tropicalis(Fig.6).

    Fig.6.Indirect immunofluorescence analysis of IgG antibodies in antisera from mice immunized with 1a–1c binding ability to C.tropicalis.Bright field (left), immunofluorescence (middle), and overlay (right).Scale bar: 50 μm.

    In summary, a series of phosphorylatedβ-1,2-mannan oligosaccharides as mimics of antigenic factor 5 were prepared in a highly convergent and effective way, which were subsequently coupled with KLH or HSA to generate corresponding glycoconjugates.The immunological properties of 1a–1c were evaluated in BALB/c mice and the ELISA resultants revealed that all KLH-conjugates could induce robust T cell-dependent responses and produce high levels ofβ-1,2-mannan specific IgG antibodies.Furthermore, these KLHconjugates displayed self-adjuvanting property that could promote robust antibody responses even in absence of an external adjuvant.Additionally, we investigated the affinity between the elicited antisera andC.albicanscells, and the immunofluorescence and flow cytometry assays clearly reflected that the order of binding affinity wasβ-1,2-(Man)3> β-1,2-(Man)2> β-1,2-(Man)4, which means that the antibody induced by 1b could react well with the oligosaccharide antigens on fungus cell and the length of linearβ-1,2-mannan had a significant impact on their antigenicity.Thus,it is concluded thatβ-1,2-(Man)3is an optimally antigenic target for designing antifungal vaccine and the semi-synthesizedβ-1,2-mannotrios–KLH conjugate 1b would be of great potential as a vaccine candidate.

    Declaration of competing interest

    We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

    Acknowledgment

    This work was financially supported by the Natural Science Foundation of China (Nos.21502223, 81773580).

    Supplementary materials

    Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.12.065.

    一二三四在线观看免费中文在| 国产综合懂色| 夜夜躁狠狠躁天天躁| 国产亚洲精品av在线| 亚洲专区国产一区二区| 女人高潮潮喷娇喘18禁视频| 欧美一区二区精品小视频在线| 亚洲中文字幕一区二区三区有码在线看 | 99国产精品99久久久久| 婷婷丁香在线五月| 免费看日本二区| 国产亚洲精品久久久com| 亚洲中文av在线| 国产乱人视频| 国产在线精品亚洲第一网站| 禁无遮挡网站| 国产精品一及| 999久久久国产精品视频| 一级毛片高清免费大全| 蜜桃久久精品国产亚洲av| 亚洲人成电影免费在线| 亚洲五月天丁香| 国产精品精品国产色婷婷| 国产精品一区二区三区四区久久| 欧美午夜高清在线| 国产不卡一卡二| 18禁国产床啪视频网站| 99riav亚洲国产免费| 成人亚洲精品av一区二区| 日韩欧美在线乱码| 久久国产乱子伦精品免费另类| 国产精品久久久久久久电影 | 亚洲av成人av| 1024手机看黄色片| 脱女人内裤的视频| 久久久久久人人人人人| 亚洲精品国产精品久久久不卡| 嫁个100分男人电影在线观看| 美女扒开内裤让男人捅视频| 老司机在亚洲福利影院| 国产91精品成人一区二区三区| 欧美激情在线99| 国产69精品久久久久777片 | 男插女下体视频免费在线播放| 精品久久久久久成人av| 老熟妇乱子伦视频在线观看| 18禁黄网站禁片免费观看直播| 亚洲狠狠婷婷综合久久图片| 97超级碰碰碰精品色视频在线观看| 天堂av国产一区二区熟女人妻| 国产极品精品免费视频能看的| 国产免费av片在线观看野外av| 91在线精品国自产拍蜜月 | 亚洲成av人片在线播放无| 久久久久久人人人人人| 久久久精品欧美日韩精品| 一级作爱视频免费观看| 一级毛片高清免费大全| 久久人人精品亚洲av| 淫秽高清视频在线观看| 夜夜夜夜夜久久久久| 看片在线看免费视频| 999久久久精品免费观看国产| 美女黄网站色视频| 亚洲成av人片免费观看| 国产激情欧美一区二区| 国产三级中文精品| 国产精品女同一区二区软件 | 国产免费av片在线观看野外av| 日日干狠狠操夜夜爽| 亚洲一区高清亚洲精品| 一区二区三区国产精品乱码| 久久国产精品影院| 国产精品一及| 一个人免费在线观看电影 | 国产又色又爽无遮挡免费看| 亚洲五月天丁香| 日本三级黄在线观看| 欧美激情久久久久久爽电影| 国产蜜桃级精品一区二区三区| 天天躁日日操中文字幕| 久久国产精品人妻蜜桃| 一区二区三区高清视频在线| or卡值多少钱| 色av中文字幕| 亚洲中文字幕日韩| 亚洲av电影在线进入| 老司机午夜十八禁免费视频| 国内精品一区二区在线观看| 中文字幕人成人乱码亚洲影| 又大又爽又粗| 国产欧美日韩精品一区二区| 无遮挡黄片免费观看| 欧美成人性av电影在线观看| 天堂影院成人在线观看| 久久久久久人人人人人| 51午夜福利影视在线观看| 成人三级做爰电影| 精品国产超薄肉色丝袜足j| 一二三四社区在线视频社区8| 麻豆成人午夜福利视频| 国产精品亚洲美女久久久| 少妇人妻一区二区三区视频| 亚洲精品久久国产高清桃花| 国产av麻豆久久久久久久| www.自偷自拍.com| 欧美性猛交╳xxx乱大交人| 99久久精品国产亚洲精品| 一本综合久久免费| 国产蜜桃级精品一区二区三区| 中国美女看黄片| 变态另类丝袜制服| 亚洲国产色片| 老司机深夜福利视频在线观看| 18禁黄网站禁片免费观看直播| 夜夜爽天天搞| 啦啦啦韩国在线观看视频| 亚洲午夜精品一区,二区,三区| 久久亚洲精品不卡| 久9热在线精品视频| 日日摸夜夜添夜夜添小说| 18禁观看日本| 91在线精品国自产拍蜜月 | 免费看a级黄色片| 制服丝袜大香蕉在线| 婷婷丁香在线五月| 国产午夜福利久久久久久| 国产精品九九99| 很黄的视频免费| 日韩国内少妇激情av| avwww免费| 又大又爽又粗| 色综合婷婷激情| 在线视频色国产色| 国产精华一区二区三区| 美女被艹到高潮喷水动态| 国产精品久久久久久亚洲av鲁大| 男女视频在线观看网站免费| 可以在线观看毛片的网站| 97碰自拍视频| 嫩草影院入口| 国产激情偷乱视频一区二区| 美女大奶头视频| 中国美女看黄片| 一级毛片女人18水好多| 精品人妻1区二区| 我要搜黄色片| 中文字幕久久专区| www日本黄色视频网| 天天躁日日操中文字幕| 中文字幕久久专区| 久久亚洲精品不卡| 国内精品久久久久久久电影| 欧美不卡视频在线免费观看| 国产精品一区二区三区四区久久| 日本黄色片子视频| 身体一侧抽搐| 亚洲精品中文字幕一二三四区| 色尼玛亚洲综合影院| 精品国产亚洲在线| 少妇裸体淫交视频免费看高清| 成熟少妇高潮喷水视频| 99久久久亚洲精品蜜臀av| 久久久精品欧美日韩精品| 精品久久久久久成人av| 亚洲av成人不卡在线观看播放网| 欧美激情在线99| 91老司机精品| 国产野战对白在线观看| 日韩欧美免费精品| 国产在线精品亚洲第一网站| 美女大奶头视频| 一本久久中文字幕| 午夜日韩欧美国产| 男女下面进入的视频免费午夜| 嫁个100分男人电影在线观看| 成人精品一区二区免费| 国内揄拍国产精品人妻在线| aaaaa片日本免费| 婷婷亚洲欧美| 一个人免费在线观看的高清视频| 亚洲天堂国产精品一区在线| 日韩成人在线观看一区二区三区| 一本一本综合久久| 曰老女人黄片| 国产一区二区在线av高清观看| 欧美黄色淫秽网站| av在线蜜桃| 我要搜黄色片| 国产av麻豆久久久久久久| 日韩 欧美 亚洲 中文字幕| 国产精华一区二区三区| 老熟妇乱子伦视频在线观看| 久久久久久久午夜电影| 中文字幕最新亚洲高清| 国产男靠女视频免费网站| 日本 欧美在线| 可以在线观看毛片的网站| 99re在线观看精品视频| 18禁黄网站禁片午夜丰满| 欧美精品啪啪一区二区三区| 欧美一区二区国产精品久久精品| 两个人看的免费小视频| 国产一区二区在线观看日韩 | 成年免费大片在线观看| 99久久国产精品久久久| 麻豆成人av在线观看| 亚洲精品国产精品久久久不卡| 午夜福利成人在线免费观看| 日韩欧美国产一区二区入口| 日本 欧美在线| 麻豆一二三区av精品| 一a级毛片在线观看| 亚洲欧美日韩高清专用| 看黄色毛片网站| 老熟妇仑乱视频hdxx| 亚洲欧美一区二区三区黑人| 亚洲精品456在线播放app | 国产亚洲av嫩草精品影院| 9191精品国产免费久久| 免费av毛片视频| 久久这里只有精品19| 国产精品一区二区精品视频观看| 99国产综合亚洲精品| 黄色丝袜av网址大全| 亚洲 国产 在线| 天天躁日日操中文字幕| 亚洲专区字幕在线| 女生性感内裤真人,穿戴方法视频| 特大巨黑吊av在线直播| 亚洲av成人不卡在线观看播放网| 久久久久国内视频| 欧美日本视频| 欧美黑人欧美精品刺激| 国产精品久久电影中文字幕| 少妇人妻一区二区三区视频| 国内精品久久久久久久电影| 亚洲成人久久性| 亚洲av熟女| 99热精品在线国产| 97超视频在线观看视频| 亚洲精品456在线播放app | 成人亚洲精品av一区二区| 久久久久久九九精品二区国产| 天天躁日日操中文字幕| 欧美精品啪啪一区二区三区| 俄罗斯特黄特色一大片| 可以在线观看毛片的网站| 男女做爰动态图高潮gif福利片| 99久久国产精品久久久| 两个人的视频大全免费| 性色avwww在线观看| 搡老熟女国产l中国老女人| 久久中文看片网| 亚洲第一欧美日韩一区二区三区| 日韩精品青青久久久久久| 麻豆av在线久日| svipshipincom国产片| 成人国产一区最新在线观看| 一本一本综合久久| 日韩中文字幕欧美一区二区| 老鸭窝网址在线观看| 麻豆一二三区av精品| 宅男免费午夜| 欧美极品一区二区三区四区| 亚洲乱码一区二区免费版| 久9热在线精品视频| 亚洲欧美精品综合一区二区三区| av视频在线观看入口| 久久久久久久久中文| 美女高潮喷水抽搐中文字幕| 国内久久婷婷六月综合欲色啪| 婷婷精品国产亚洲av在线| 午夜免费激情av| 久久久久国产精品人妻aⅴ院| 国产一区二区激情短视频| 亚洲男人的天堂狠狠| 人人妻人人澡欧美一区二区| 亚洲人成伊人成综合网2020| 一区二区三区高清视频在线| 精品欧美国产一区二区三| 成年版毛片免费区| cao死你这个sao货| 亚洲最大成人中文| 日韩欧美免费精品| 一本综合久久免费| 国产精品女同一区二区软件 | 免费人成视频x8x8入口观看| 中文资源天堂在线| 亚洲av日韩精品久久久久久密| 男女那种视频在线观看| 国产私拍福利视频在线观看| 午夜成年电影在线免费观看| 精品国产三级普通话版| 美女扒开内裤让男人捅视频| 少妇丰满av| 首页视频小说图片口味搜索| 日本五十路高清| 1024手机看黄色片| 我的老师免费观看完整版| 嫩草影院入口| 免费av不卡在线播放| 国产黄色小视频在线观看| 真人做人爱边吃奶动态| АⅤ资源中文在线天堂| 精品一区二区三区av网在线观看| 色吧在线观看| 在线播放国产精品三级| 色综合亚洲欧美另类图片| 老司机福利观看| 色在线成人网| 夜夜夜夜夜久久久久| 亚洲 国产 在线| 男人和女人高潮做爰伦理| 国产精品亚洲一级av第二区| 18禁裸乳无遮挡免费网站照片| 成人高潮视频无遮挡免费网站| 99国产综合亚洲精品| 国产1区2区3区精品| 久久久久久久久久黄片| 精品久久久久久久久久久久久| av欧美777| 婷婷丁香在线五月| 国产不卡一卡二| 又爽又黄无遮挡网站| 神马国产精品三级电影在线观看| 狂野欧美白嫩少妇大欣赏| 悠悠久久av| 高清毛片免费观看视频网站| 日本黄色视频三级网站网址| 十八禁人妻一区二区| 亚洲专区国产一区二区| 久久亚洲真实| 在线观看66精品国产| 国产成人精品久久二区二区91| 熟妇人妻久久中文字幕3abv| 免费大片18禁| 亚洲一区二区三区不卡视频| 嫩草影院精品99| 久久久久久久久中文| 国产亚洲欧美在线一区二区| 日韩欧美在线乱码| 偷拍熟女少妇极品色| 人人妻人人看人人澡| 成人特级av手机在线观看| 国产欧美日韩精品一区二区| 色综合站精品国产| 波多野结衣巨乳人妻| 老汉色av国产亚洲站长工具| 国产精品野战在线观看| 国产一区二区激情短视频| 国产精品电影一区二区三区| 色综合站精品国产| 91在线观看av| 黄频高清免费视频| 亚洲午夜精品一区,二区,三区| 国产高清有码在线观看视频| 国内精品一区二区在线观看| 久久亚洲真实| 在线观看免费视频日本深夜| 国产成人影院久久av| 国产视频一区二区在线看| 一边摸一边抽搐一进一小说| 久久精品91蜜桃| 国产成人影院久久av| 九九在线视频观看精品| 禁无遮挡网站| 国产精品综合久久久久久久免费| 熟女人妻精品中文字幕| av天堂在线播放| 中文字幕精品亚洲无线码一区| 日韩欧美 国产精品| 国产真实乱freesex| 三级国产精品欧美在线观看 | 最好的美女福利视频网| 欧美丝袜亚洲另类 | 亚洲国产中文字幕在线视频| 婷婷六月久久综合丁香| 九色国产91popny在线| 岛国视频午夜一区免费看| 日本一本二区三区精品| 国产一区二区在线av高清观看| 国产1区2区3区精品| 90打野战视频偷拍视频| 搞女人的毛片| 国产成人精品久久二区二区91| 曰老女人黄片| 俺也久久电影网| 精品国产超薄肉色丝袜足j| 一本一本综合久久| 欧美+亚洲+日韩+国产| 国产麻豆成人av免费视频| 男人舔女人的私密视频| 亚洲成a人片在线一区二区| 午夜免费成人在线视频| 91麻豆av在线| 婷婷六月久久综合丁香| 国产精品一区二区免费欧美| 国产一区二区激情短视频| 亚洲中文日韩欧美视频| 偷拍熟女少妇极品色| 老司机深夜福利视频在线观看| 日日干狠狠操夜夜爽| 老司机在亚洲福利影院| 国产精华一区二区三区| 日韩人妻高清精品专区| 亚洲一区高清亚洲精品| 久久亚洲精品不卡| 国产精品一区二区免费欧美| 成年女人永久免费观看视频| 国产伦一二天堂av在线观看| 99久久无色码亚洲精品果冻| 动漫黄色视频在线观看| 成人性生交大片免费视频hd| 最好的美女福利视频网| 精品99又大又爽又粗少妇毛片 | 99热精品在线国产| 亚洲性夜色夜夜综合| 一本综合久久免费| 老鸭窝网址在线观看| 久久香蕉精品热| 后天国语完整版免费观看| 日韩欧美 国产精品| 精品一区二区三区四区五区乱码| 日韩欧美精品v在线| 欧美另类亚洲清纯唯美| 亚洲av免费在线观看| 中文字幕久久专区| 欧美性猛交╳xxx乱大交人| 亚洲欧美日韩东京热| 欧美在线一区亚洲| 亚洲人成网站在线播放欧美日韩| 成年人黄色毛片网站| 日韩人妻高清精品专区| 亚洲精品456在线播放app | 人人妻人人看人人澡| 制服丝袜大香蕉在线| 国产乱人伦免费视频| 日本一二三区视频观看| ponron亚洲| e午夜精品久久久久久久| 91av网一区二区| 丰满人妻熟妇乱又伦精品不卡| 久久中文看片网| 美女被艹到高潮喷水动态| 亚洲性夜色夜夜综合| www日本在线高清视频| 亚洲五月天丁香| 五月伊人婷婷丁香| 国产成人啪精品午夜网站| 欧美黄色片欧美黄色片| 色综合婷婷激情| 亚洲精品中文字幕一二三四区| 天堂√8在线中文| 久久久精品大字幕| 国产精品精品国产色婷婷| 成人国产综合亚洲| 男人和女人高潮做爰伦理| www.自偷自拍.com| 欧美日韩亚洲国产一区二区在线观看| 香蕉av资源在线| 高潮久久久久久久久久久不卡| 久久久久久大精品| 欧美绝顶高潮抽搐喷水| 中国美女看黄片| 午夜视频精品福利| 欧美3d第一页| 国产蜜桃级精品一区二区三区| 人妻丰满熟妇av一区二区三区| 成人特级av手机在线观看| 99国产精品一区二区三区| 中亚洲国语对白在线视频| 成年女人看的毛片在线观看| xxxwww97欧美| 国产精品av视频在线免费观看| xxx96com| 久久中文字幕人妻熟女| 小说图片视频综合网站| 国产成人一区二区三区免费视频网站| 一a级毛片在线观看| 亚洲人成网站高清观看| or卡值多少钱| 亚洲欧美精品综合一区二区三区| 熟女人妻精品中文字幕| 中文字幕人成人乱码亚洲影| 人妻丰满熟妇av一区二区三区| 99国产综合亚洲精品| 国产精品1区2区在线观看.| 香蕉国产在线看| 神马国产精品三级电影在线观看| 日韩欧美在线二视频| 哪里可以看免费的av片| 日本熟妇午夜| 真人一进一出gif抽搐免费| 人人妻,人人澡人人爽秒播| 成熟少妇高潮喷水视频| 长腿黑丝高跟| 亚洲专区字幕在线| 国产精华一区二区三区| 午夜影院日韩av| 日本三级黄在线观看| 国产一区二区在线av高清观看| 两性午夜刺激爽爽歪歪视频在线观看| 久久性视频一级片| 免费看a级黄色片| 久久久久精品国产欧美久久久| 国产精品日韩av在线免费观看| 久久久久国产精品人妻aⅴ院| 欧美成狂野欧美在线观看| 村上凉子中文字幕在线| 精品久久久久久久久久久久久| 色综合站精品国产| 国产精品98久久久久久宅男小说| 狂野欧美白嫩少妇大欣赏| 最新中文字幕久久久久 | 久久99热这里只有精品18| 天堂av国产一区二区熟女人妻| 成人午夜高清在线视频| 日韩欧美 国产精品| 特大巨黑吊av在线直播| 亚洲精品美女久久av网站| 亚洲最大成人中文| 麻豆成人午夜福利视频| 一a级毛片在线观看| 99精品在免费线老司机午夜| 亚洲aⅴ乱码一区二区在线播放| 国产一级毛片七仙女欲春2| xxxwww97欧美| 97超视频在线观看视频| 熟妇人妻久久中文字幕3abv| tocl精华| 国产成人系列免费观看| 一级毛片女人18水好多| 亚洲av熟女| 国产高清视频在线播放一区| 亚洲avbb在线观看| 亚洲av日韩精品久久久久久密| 日韩精品中文字幕看吧| 可以在线观看的亚洲视频| 99re在线观看精品视频| 国产av麻豆久久久久久久| 色播亚洲综合网| 大型黄色视频在线免费观看| 亚洲第一电影网av| 欧美性猛交╳xxx乱大交人| 国产精品久久久久久久电影 | www日本在线高清视频| 精品一区二区三区视频在线 | 亚洲精品中文字幕一二三四区| 我的老师免费观看完整版| 成熟少妇高潮喷水视频| 久久久久久久午夜电影| 又紧又爽又黄一区二区| 亚洲欧美激情综合另类| 日韩欧美三级三区| 欧美乱色亚洲激情| 午夜精品在线福利| 男人舔女人下体高潮全视频| 少妇的逼水好多| 99riav亚洲国产免费| 亚洲成人精品中文字幕电影| 高潮久久久久久久久久久不卡| 国产成人精品久久二区二区91| 精品电影一区二区在线| 高清在线国产一区| 噜噜噜噜噜久久久久久91| 中文亚洲av片在线观看爽| av片东京热男人的天堂| 亚洲,欧美精品.| 欧美乱色亚洲激情| 午夜精品久久久久久毛片777| 国产真人三级小视频在线观看| 国产精品野战在线观看| 亚洲精品久久国产高清桃花| 无遮挡黄片免费观看| 一级毛片女人18水好多| 嫩草影院入口| 国产野战对白在线观看| 一级毛片女人18水好多| 嫩草影院入口| 亚洲乱码一区二区免费版| 精品国产乱子伦一区二区三区| а√天堂www在线а√下载| 每晚都被弄得嗷嗷叫到高潮| 99热这里只有精品一区 | 高潮久久久久久久久久久不卡| 69av精品久久久久久| 国产精品影院久久| 久久国产精品人妻蜜桃| 国产成人精品无人区| 亚洲专区中文字幕在线| 精品午夜福利视频在线观看一区| 中文字幕最新亚洲高清| 露出奶头的视频| 一二三四在线观看免费中文在| 欧美黄色淫秽网站| 床上黄色一级片| 亚洲精品一卡2卡三卡4卡5卡| 色视频www国产| 九色成人免费人妻av| 精品不卡国产一区二区三区| 欧美中文日本在线观看视频| 亚洲熟妇中文字幕五十中出| 国产精品98久久久久久宅男小说| 国产亚洲欧美98| 中文字幕人妻丝袜一区二区| 成人国产综合亚洲| 欧美色欧美亚洲另类二区| 长腿黑丝高跟| 欧美日韩综合久久久久久 | 欧美一区二区精品小视频在线| 亚洲欧洲精品一区二区精品久久久| 精品免费久久久久久久清纯| ponron亚洲| 观看免费一级毛片|