• <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.

    在线观看免费视频网站a站| 免费大片18禁| 久久人人爽人人片av| 欧美日韩精品成人综合77777| 乱码一卡2卡4卡精品| 久久午夜综合久久蜜桃| 国产精品一区二区在线观看99| 大香蕉97超碰在线| 免费人妻精品一区二区三区视频| 亚洲国产毛片av蜜桃av| 9色porny在线观看| 国产女主播在线喷水免费视频网站| 麻豆精品久久久久久蜜桃| 国产成人av激情在线播放| 波野结衣二区三区在线| 啦啦啦啦在线视频资源| 97在线视频观看| 人成视频在线观看免费观看| 青春草视频在线免费观看| xxxhd国产人妻xxx| 国产精品一国产av| 最新的欧美精品一区二区| 国产av精品麻豆| 久久毛片免费看一区二区三区| 嫩草影院入口| 哪个播放器可以免费观看大片| 免费在线观看完整版高清| 国产精品国产三级国产专区5o| 日本猛色少妇xxxxx猛交久久| 1024视频免费在线观看| 午夜免费男女啪啪视频观看| 久久国内精品自在自线图片| 精品国产一区二区久久| 国产在线免费精品| 国产一级毛片在线| 一级a做视频免费观看| 欧美精品人与动牲交sv欧美| 久久国产精品男人的天堂亚洲 | 亚洲精品日本国产第一区| 18在线观看网站| 国产一区有黄有色的免费视频| 亚洲国产最新在线播放| 亚洲国产av新网站| 性高湖久久久久久久久免费观看| 欧美97在线视频| 黄色视频在线播放观看不卡| 亚洲欧洲精品一区二区精品久久久 | 国国产精品蜜臀av免费| 美女脱内裤让男人舔精品视频| 国产无遮挡羞羞视频在线观看| 国产精品欧美亚洲77777| 日本免费在线观看一区| 在线观看一区二区三区激情| 久久精品国产综合久久久 | 成人18禁高潮啪啪吃奶动态图| 精品福利永久在线观看| 久久久久久久亚洲中文字幕| 亚洲av免费高清在线观看| 国产激情久久老熟女| 日韩av在线免费看完整版不卡| 热99久久久久精品小说推荐| 97在线视频观看| 亚洲欧美色中文字幕在线| 女人精品久久久久毛片| 人人妻人人爽人人添夜夜欢视频| 久久人人爽人人爽人人片va| 日韩视频在线欧美| 在线观看三级黄色| 亚洲欧美色中文字幕在线| 黑人高潮一二区| 巨乳人妻的诱惑在线观看| 最近最新中文字幕免费大全7| 天天躁夜夜躁狠狠躁躁| 国产麻豆69| 欧美变态另类bdsm刘玥| 一边摸一边做爽爽视频免费| 制服人妻中文乱码| 国产一区二区三区av在线| 99国产精品免费福利视频| 中文字幕亚洲精品专区| 99久国产av精品国产电影| 午夜免费鲁丝| 亚洲综合色惰| 国产极品天堂在线| videosex国产| 国产av一区二区精品久久| 欧美成人精品欧美一级黄| 在线免费观看不下载黄p国产| 久久 成人 亚洲| 亚洲欧洲日产国产| 99热国产这里只有精品6| 母亲3免费完整高清在线观看 | 大香蕉97超碰在线| xxxhd国产人妻xxx| 中国三级夫妇交换| 搡女人真爽免费视频火全软件| 亚洲经典国产精华液单| 免费少妇av软件| 国产成人aa在线观看| 免费大片18禁| 亚洲欧洲精品一区二区精品久久久 | 国产探花极品一区二区| 十八禁高潮呻吟视频| 超碰97精品在线观看| 91精品伊人久久大香线蕉| 美女大奶头黄色视频| 晚上一个人看的免费电影| 成人国产av品久久久| 国产精品人妻久久久久久| 亚洲内射少妇av| 日韩成人伦理影院| 精品少妇久久久久久888优播| 久久久久久久久久久免费av| 日本欧美国产在线视频| 日韩人妻精品一区2区三区| 国产精品蜜桃在线观看| 日日撸夜夜添| 最近中文字幕2019免费版| 高清视频免费观看一区二区| 午夜福利乱码中文字幕| 国产精品久久久久久久久免| 下体分泌物呈黄色| 日韩欧美精品免费久久| 青春草亚洲视频在线观看| 久久这里只有精品19| 亚洲欧美精品自产自拍| 欧美激情极品国产一区二区三区 | 亚洲激情五月婷婷啪啪| 免费高清在线观看日韩| 高清视频免费观看一区二区| 日韩精品免费视频一区二区三区 | 午夜福利乱码中文字幕| 欧美成人午夜免费资源| 如何舔出高潮| 热re99久久精品国产66热6| 亚洲欧美日韩另类电影网站| 一本大道久久a久久精品| 丝袜美足系列| 高清av免费在线| 亚洲国产欧美在线一区| 国产麻豆69| 搡女人真爽免费视频火全软件| 两性夫妻黄色片 | 我的女老师完整版在线观看| 国产成人免费观看mmmm| 亚洲国产精品专区欧美| 波多野结衣一区麻豆| 18在线观看网站| 最新的欧美精品一区二区| 国产永久视频网站| 国产av精品麻豆| 夫妻性生交免费视频一级片| 国产成人精品福利久久| 九草在线视频观看| 91精品三级在线观看| a级片在线免费高清观看视频| kizo精华| 亚洲,欧美,日韩| 热99久久久久精品小说推荐| 亚洲精品一二三| 成年动漫av网址| 大片电影免费在线观看免费| 90打野战视频偷拍视频| 欧美成人精品欧美一级黄| 成人漫画全彩无遮挡| 国产福利在线免费观看视频| 成人午夜精彩视频在线观看| 极品少妇高潮喷水抽搐| 亚洲av免费高清在线观看| 日韩制服骚丝袜av| 性高湖久久久久久久久免费观看| 老女人水多毛片| 国产av国产精品国产| 丝袜人妻中文字幕| 亚洲三级黄色毛片| 亚洲欧美清纯卡通| 国产精品秋霞免费鲁丝片| 成年人免费黄色播放视频| 下体分泌物呈黄色| www.色视频.com| 免费大片黄手机在线观看| 久久人人爽人人片av| 成人无遮挡网站| 亚洲成人手机| 久久精品久久精品一区二区三区| 男女国产视频网站| 国产亚洲午夜精品一区二区久久| 如日韩欧美国产精品一区二区三区| 亚洲成av片中文字幕在线观看 | 欧美精品一区二区免费开放| 日韩av在线免费看完整版不卡| 亚洲精品视频女| 亚洲精品视频女| 亚洲av国产av综合av卡| 日本黄大片高清| 女性生殖器流出的白浆| 高清视频免费观看一区二区| 亚洲人成网站在线观看播放| 精品视频人人做人人爽| 久久精品熟女亚洲av麻豆精品| 精品亚洲乱码少妇综合久久| 九九爱精品视频在线观看| 欧美成人精品欧美一级黄| 欧美亚洲日本最大视频资源| 欧美成人午夜精品| 国产在视频线精品| 欧美国产精品va在线观看不卡| 王馨瑶露胸无遮挡在线观看| 精品第一国产精品| 精品一区在线观看国产| 亚洲综合色惰| 国产麻豆69| 天天操日日干夜夜撸| 亚洲欧美一区二区三区黑人 | 久久狼人影院| 青春草视频在线免费观看| 老女人水多毛片| 成年人午夜在线观看视频| 日韩免费高清中文字幕av| 精品卡一卡二卡四卡免费| 午夜老司机福利剧场| 国产精品嫩草影院av在线观看| 美女内射精品一级片tv| 校园人妻丝袜中文字幕| 91精品伊人久久大香线蕉| 看免费av毛片| 高清不卡的av网站| 国产1区2区3区精品| 亚洲色图综合在线观看| 91精品伊人久久大香线蕉| 亚洲国产最新在线播放| 国产精品久久久久久av不卡| 久久精品国产综合久久久 | 中文字幕精品免费在线观看视频 | 九草在线视频观看| 免费大片黄手机在线观看| 亚洲精品美女久久av网站| 看免费成人av毛片| 一区二区三区精品91| 国产精品三级大全| 精品久久久久久电影网| 寂寞人妻少妇视频99o| 国产精品一国产av| 成人国语在线视频| 丰满迷人的少妇在线观看| 啦啦啦视频在线资源免费观看| 国产日韩欧美亚洲二区| 中文天堂在线官网| 日本爱情动作片www.在线观看| 国产精品蜜桃在线观看| 亚洲国产日韩一区二区| 尾随美女入室| 亚洲精品成人av观看孕妇| 午夜老司机福利剧场| 在现免费观看毛片| 午夜激情av网站| 在线观看www视频免费| 久久久久久人人人人人| 国产亚洲av片在线观看秒播厂| 午夜影院在线不卡| 一区二区日韩欧美中文字幕 | 国产淫语在线视频| 国产又色又爽无遮挡免| 久久久精品免费免费高清| 亚洲国产精品999| 黑丝袜美女国产一区| 欧美xxⅹ黑人| 精品国产乱码久久久久久小说| 国产精品免费大片| 国产亚洲最大av| 看免费av毛片| 最后的刺客免费高清国语| 黄色视频在线播放观看不卡| 青春草国产在线视频| 免费看av在线观看网站| 久久人人97超碰香蕉20202| 国产成人精品一,二区| 午夜视频国产福利| 欧美人与善性xxx| 国产不卡av网站在线观看| 99国产精品免费福利视频| 大香蕉久久网| 日本-黄色视频高清免费观看| 最近最新中文字幕大全免费视频 | 欧美日本中文国产一区发布| 国产一区二区三区综合在线观看 | 黑人猛操日本美女一级片| 天堂8中文在线网| 最近中文字幕高清免费大全6| 亚洲婷婷狠狠爱综合网| 国产老妇伦熟女老妇高清| 男人舔女人的私密视频| 精品国产一区二区三区四区第35| 精品人妻在线不人妻| 精品国产一区二区三区四区第35| 亚洲国产精品999| 成人免费观看视频高清| 黑丝袜美女国产一区| 视频在线观看一区二区三区| 男女边摸边吃奶| 国产熟女午夜一区二区三区| 日韩中文字幕视频在线看片| 久久久久人妻精品一区果冻| 亚洲av电影在线观看一区二区三区| av卡一久久| 国产69精品久久久久777片| 亚洲av在线观看美女高潮| 成年动漫av网址| 亚洲成av片中文字幕在线观看 | 一区二区三区四区激情视频| av电影中文网址| 人妻系列 视频| 免费少妇av软件| 色94色欧美一区二区| 日韩av在线免费看完整版不卡| 国产一区二区在线观看日韩| 精品一区二区三区四区五区乱码 | 久久久精品免费免费高清| 欧美+日韩+精品| 在线天堂最新版资源| 久久99精品国语久久久| 一级毛片电影观看| 免费日韩欧美在线观看| 亚洲伊人色综图| 成年人免费黄色播放视频| 成人毛片a级毛片在线播放| 七月丁香在线播放| 啦啦啦中文免费视频观看日本| 黄色视频在线播放观看不卡| 国产精品久久久av美女十八| 亚洲av电影在线进入| 日本av免费视频播放| 有码 亚洲区| 大香蕉97超碰在线| 乱码一卡2卡4卡精品| 亚洲在久久综合| 日韩一本色道免费dvd| 亚洲av.av天堂| 亚洲,一卡二卡三卡| 久久99热6这里只有精品| 丝袜喷水一区| 午夜免费男女啪啪视频观看| 一级,二级,三级黄色视频| 视频中文字幕在线观看| 国产精品嫩草影院av在线观看| av黄色大香蕉| 九草在线视频观看| 最黄视频免费看| 久久午夜综合久久蜜桃| 亚洲国产精品成人久久小说| 新久久久久国产一级毛片| 90打野战视频偷拍视频| 午夜av观看不卡| 一级片'在线观看视频| 午夜91福利影院| 蜜桃国产av成人99| kizo精华| 欧美精品一区二区大全| 国产国拍精品亚洲av在线观看| 夜夜骑夜夜射夜夜干| 国产日韩欧美亚洲二区| 90打野战视频偷拍视频| 国产国语露脸激情在线看| 免费观看性生交大片5| 一边摸一边做爽爽视频免费| 久久精品熟女亚洲av麻豆精品| 熟女电影av网| 日本wwww免费看| 少妇猛男粗大的猛烈进出视频| 成人18禁高潮啪啪吃奶动态图| 啦啦啦视频在线资源免费观看| 女人被躁到高潮嗷嗷叫费观| 国产探花极品一区二区| 国产一区二区三区av在线| 观看av在线不卡| 黑人猛操日本美女一级片| 日韩免费高清中文字幕av| av线在线观看网站| 亚洲人成77777在线视频| 国产精品秋霞免费鲁丝片| 少妇猛男粗大的猛烈进出视频| 国产深夜福利视频在线观看| 国产毛片在线视频| 亚洲精品,欧美精品| xxxhd国产人妻xxx| 啦啦啦啦在线视频资源| 日韩成人伦理影院| 在线观看免费日韩欧美大片| 两个人看的免费小视频| 亚洲五月色婷婷综合| 免费看不卡的av| 日产精品乱码卡一卡2卡三| 久久午夜综合久久蜜桃| 激情视频va一区二区三区| 国产成人一区二区在线| 免费人妻精品一区二区三区视频| 男女边吃奶边做爰视频| 国产老妇伦熟女老妇高清| 青春草视频在线免费观看| 男女啪啪激烈高潮av片| 九色成人免费人妻av| 热re99久久国产66热| 欧美另类一区| 22中文网久久字幕| 久久人人97超碰香蕉20202| 精品久久国产蜜桃| 国产成人精品福利久久| 老司机影院毛片| 99香蕉大伊视频| 91在线精品国自产拍蜜月| 国产69精品久久久久777片| 午夜福利,免费看| 91午夜精品亚洲一区二区三区| 国产精品欧美亚洲77777| 亚洲精品日本国产第一区| 精品人妻在线不人妻| 男的添女的下面高潮视频| 亚洲欧洲精品一区二区精品久久久 | 亚洲欧美精品自产自拍| 日韩伦理黄色片| 久久久久久久精品精品| 国产精品国产三级国产专区5o| 午夜免费男女啪啪视频观看| 国产永久视频网站| 午夜久久久在线观看| 国产片特级美女逼逼视频| 日韩一区二区视频免费看| 亚洲欧美精品自产自拍| 又粗又硬又长又爽又黄的视频| 高清欧美精品videossex| 亚洲三级黄色毛片| 一级片免费观看大全| 欧美另类一区| 黄色 视频免费看| 欧美日本中文国产一区发布| 亚洲色图 男人天堂 中文字幕 | av国产久精品久网站免费入址| 久久精品国产亚洲av天美| 亚洲综合色网址| 国产成人av激情在线播放| 丝袜在线中文字幕| 亚洲成人av在线免费| 国国产精品蜜臀av免费| 欧美丝袜亚洲另类| 色网站视频免费| 26uuu在线亚洲综合色| 久久国内精品自在自线图片| 成人午夜精彩视频在线观看| 日韩中字成人| 下体分泌物呈黄色| 欧美日韩综合久久久久久| 黑人欧美特级aaaaaa片| 天天躁夜夜躁狠狠躁躁| av网站免费在线观看视频| 狂野欧美激情性xxxx在线观看| 熟女人妻精品中文字幕| 青春草视频在线免费观看| 波多野结衣一区麻豆| 我要看黄色一级片免费的| 宅男免费午夜| 亚洲av男天堂| 亚洲精品成人av观看孕妇| 免费av不卡在线播放| 国产亚洲午夜精品一区二区久久| 亚洲精品国产av蜜桃| 母亲3免费完整高清在线观看 | 亚洲一区二区三区欧美精品| 中文字幕av电影在线播放| 免费人成在线观看视频色| 少妇被粗大猛烈的视频| 丝袜脚勾引网站| 大陆偷拍与自拍| 成人18禁高潮啪啪吃奶动态图| 精品亚洲成a人片在线观看| 只有这里有精品99| av不卡在线播放| 国产在线视频一区二区| 免费不卡的大黄色大毛片视频在线观看| 丰满饥渴人妻一区二区三| 一级毛片黄色毛片免费观看视频| 欧美日韩视频高清一区二区三区二| 亚洲成国产人片在线观看| 久久久久国产精品人妻一区二区| 国产精品.久久久| 国国产精品蜜臀av免费| 乱人伦中国视频| 这个男人来自地球电影免费观看 | videosex国产| 精品一区在线观看国产| 国产在线一区二区三区精| 亚洲av中文av极速乱| 精品久久久精品久久久| 久久精品国产亚洲av涩爱| 一级片'在线观看视频| 久久久久久久国产电影| 久久免费观看电影| 亚洲欧美清纯卡通| 亚洲精品美女久久久久99蜜臀 | av免费在线看不卡| 久久人人爽人人片av| 人体艺术视频欧美日本| 精品第一国产精品| 国产色婷婷99| 亚洲美女视频黄频| 欧美3d第一页| 国产永久视频网站| 精品久久久精品久久久| 不卡视频在线观看欧美| 国产男女内射视频| 丝袜喷水一区| 国产一区二区在线观看日韩| 一级黄片播放器| 国产女主播在线喷水免费视频网站| 美女内射精品一级片tv| 欧美老熟妇乱子伦牲交| 一边亲一边摸免费视频| av在线app专区| 99香蕉大伊视频| 国产乱来视频区| 国产女主播在线喷水免费视频网站| 大片免费播放器 马上看| 亚洲在久久综合| 国产高清国产精品国产三级| 国产淫语在线视频| 亚洲国产精品专区欧美| 欧美 日韩 精品 国产| 免费人妻精品一区二区三区视频| a 毛片基地| 亚洲第一区二区三区不卡| 91精品伊人久久大香线蕉| 午夜福利,免费看| 男女边吃奶边做爰视频| 亚洲欧美色中文字幕在线| 国产国语露脸激情在线看| 亚洲av综合色区一区| 日韩av不卡免费在线播放| 欧美人与性动交α欧美软件 | 国产综合精华液| 少妇被粗大的猛进出69影院 | 午夜福利影视在线免费观看| 女性生殖器流出的白浆| 综合色丁香网| kizo精华| 丰满乱子伦码专区| 国产淫语在线视频| 日韩三级伦理在线观看| 乱人伦中国视频| 国产 一区精品| 国产精品国产三级国产专区5o| 亚洲第一区二区三区不卡| 亚洲国产最新在线播放| 久久鲁丝午夜福利片| 免费大片18禁| 亚洲人成网站在线观看播放| 日本猛色少妇xxxxx猛交久久| 国产又爽黄色视频| www.色视频.com| 久久久久久人妻| 亚洲欧洲国产日韩| 在线亚洲精品国产二区图片欧美| 中文欧美无线码| 三级国产精品片| a级毛片在线看网站| 国产精品嫩草影院av在线观看| 全区人妻精品视频| 精品卡一卡二卡四卡免费| 亚洲精品美女久久久久99蜜臀 | 在线观看国产h片| 国产av一区二区精品久久| 日本猛色少妇xxxxx猛交久久| 男人添女人高潮全过程视频| 亚洲成国产人片在线观看| 91成人精品电影| 亚洲伊人久久精品综合| 亚洲精品一区蜜桃| 菩萨蛮人人尽说江南好唐韦庄| 免费黄网站久久成人精品| 亚洲欧美成人综合另类久久久| 蜜桃国产av成人99| 久久99蜜桃精品久久| 天堂俺去俺来也www色官网| 丝袜人妻中文字幕| av女优亚洲男人天堂| 中文天堂在线官网| 9热在线视频观看99| 一级片免费观看大全| 欧美日韩av久久| 国产精品 国内视频| 欧美国产精品va在线观看不卡| 欧美xxⅹ黑人| 成年人午夜在线观看视频| 国产女主播在线喷水免费视频网站| 久久久久网色| a级毛片在线看网站| 亚洲五月色婷婷综合| 日产精品乱码卡一卡2卡三| 三级国产精品片| 91国产中文字幕| 晚上一个人看的免费电影| 人人妻人人澡人人看| 亚洲精品国产av蜜桃| 日韩制服骚丝袜av| tube8黄色片| 国产精品免费大片| h视频一区二区三区| 欧美精品av麻豆av| 深夜精品福利| 日韩成人av中文字幕在线观看| 欧美激情极品国产一区二区三区 | 欧美激情国产日韩精品一区| 久久久久久久精品精品| 伦理电影大哥的女人| 精品国产国语对白av| 大话2 男鬼变身卡| 99久久综合免费|