鐮形扇頭蜱半胱氨酸蛋白酶的鑒定及其在唾液腺細(xì)胞凋亡中的作用研究
發(fā)布時(shí)間：2018-08-11 12:25
【摘要】：半胱氨酸蛋白酶是一類古老而又保守的蛋白酶,其分布廣泛,涵蓋范圍從病毒、細(xì)菌到哺乳動(dòng)物的各個(gè)組織器官,參與胚胎發(fā)育,抗原呈遞,細(xì)胞凋亡和穩(wěn)態(tài)維持等多種重要的生理活動(dòng)。作為專性體表寄生蟲,蜱以吸血宿主血液為生,在其吸血過(guò)程中可傳播細(xì)菌、真菌、病毒、支原體、原蟲等多種病原體,是重要的人畜共患病傳播媒介。唾液腺是宿主血液進(jìn)入蜱體內(nèi)最先接觸的組織器官,也是蜱體內(nèi)病原體傳播到宿主體內(nèi)的通道,因此蜱唾液腺重要分子的研究對(duì)于蜱及蜱傳病控制十分重要;隨著吸血過(guò)程的完成,蜱的唾液腺逐漸萎縮,這一現(xiàn)象是由細(xì)胞凋亡引起的,但分子機(jī)制不清。本研究以蜱的唾液腺中半胱氨酸蛋白酶分子群的鑒定為切入點(diǎn),探索了半胱氨酸蛋白酶在唾液腺發(fā)育與凋亡中的作用機(jī)制。以鐮形扇頭蜱為研究對(duì)象,對(duì)吸血前、后唾液腺轉(zhuǎn)錄組進(jìn)行測(cè)序及差異分析,分別測(cè)得上調(diào)基因1179個(gè),下調(diào)基因574個(gè),其中總轉(zhuǎn)錄庫(kù)中預(yù)測(cè)的半胱氨酸蛋白酶表達(dá)序列標(biāo)簽(EST)有25個(gè)(13個(gè)上調(diào)基因EST,1個(gè)下調(diào)基因EST);經(jīng)過(guò)基因克隆,共獲得6個(gè)半胱氨酸蛋白酶全長(zhǎng)序列,根據(jù)基因結(jié)構(gòu)和生物信息學(xué)分析,分別命名為組織蛋白酶B(CATB)、組織蛋白酶L(CATL)、半胱天冬氨酸蛋白酶-1(CASP1)、半胱天冬氨酸蛋白酶-8(CASP8)、自噬相關(guān)蛋白酶4B(ATG4B)和自噬相關(guān)蛋白酶4D(ATG4D)。通過(guò)Real-time PCR方法檢測(cè)了6種半胱氨酸蛋白酶在蜱不同發(fā)育階段和不同吸血狀態(tài)的轉(zhuǎn)錄水平,結(jié)果顯示幼蜱和若蜱吸血后所有蛋白酶的轉(zhuǎn)錄量均顯著性下調(diào),而在成蜱則全部顯著性上調(diào);6種蛋白酶在成蜱唾液腺中吸血后轉(zhuǎn)錄量上調(diào),尤其是CATB和CATL,上調(diào)差異非常顯著,而在其他組織器官中轉(zhuǎn)錄水平各有不同。對(duì)5種半胱氨酸蛋白酶進(jìn)行了體外重組蛋白表達(dá)和純化(CASP8未表達(dá)),并分別制備小鼠特異抗血清。重組蛋白的酶活實(shí)驗(yàn)顯示,各蛋白在適度濃度和PH范圍內(nèi)均表現(xiàn)一定的活性:CASP1的最適PH為5.5,可達(dá)陽(yáng)性對(duì)照酶活性的60%;組織蛋白酶B和L隨著濃度的增加,其活性受到抑制,其中組織蛋白酶L包含一個(gè)60個(gè)氨基酸的抑制結(jié)構(gòu)域,將其切除后可明顯提高組織蛋白酶L的活性,推測(cè)抑制結(jié)構(gòu)域是限制組織蛋白酶L活性的重要因素,而組織蛋白酶B的高濃度抑制效應(yīng)可能來(lái)自酶與底物的競(jìng)爭(zhēng);ATG4B和ATG4D對(duì)CASP1特異性底物具有一定活性,可達(dá)陽(yáng)性對(duì)照的40%,ATG4D對(duì)組織蛋白酶特異性底物具有良好活性,高濃度下也出現(xiàn)抑制效應(yīng)。5種重組蛋白的特異性抗血清,可分別識(shí)別成蜱體內(nèi)天然蛋白酶,其中兩種組織蛋白酶CATB和CATL在35~40 kDa之間均有2條帶,可能為酶原和激活態(tài),而自噬相關(guān)蛋白酶天然蛋白大小分別為~36 kDa(ATG4B)和~40 kDa(ATG4D),CASP1天然蛋白大小約為37 kDa,在70 kDa左右有類似二聚體結(jié)構(gòu),可能是其真正的活性態(tài)。利用免疫組化技術(shù),觀察5種半胱氨酸蛋白酶在唾液腺和腸腔中的定位,結(jié)果顯示在唾液腺中5種蛋白酶分布廣泛,而在中腸只可見ATG4D的少量分布。利用RNA干擾技術(shù),通過(guò)電轉(zhuǎn)化將合成的特異性雙鏈RNA導(dǎo)入鐮形扇頭蜱若蜱體內(nèi),對(duì)6種半胱氨酸蛋白酶分別進(jìn)行單一干擾實(shí)驗(yàn),檢測(cè)6種半胱氨酸蛋白酶之間是否存在相互作用關(guān)系,結(jié)果顯示任一蛋白酶受到抑制均可引起其他5種蛋白酶轉(zhuǎn)錄水平發(fā)生變化,特別是當(dāng)CATB和CATL轉(zhuǎn)錄受抑制可引起ATG4B和CASP1轉(zhuǎn)錄水平顯著性上調(diào),而CASP8和ATG4D轉(zhuǎn)錄受抑制時(shí)也引起CASP1轉(zhuǎn)錄水平顯著上調(diào);當(dāng)CASP1受到抑制后,CATB的轉(zhuǎn)錄水平下調(diào)顯著,ATG4D、ATG4B和CASP8的轉(zhuǎn)錄水平也受到明顯抑制,但CATL的轉(zhuǎn)錄水平未受到影響,推測(cè)CASP1對(duì)于CASP8,CATB,ATG4B和ATG4D可能具有上游調(diào)節(jié)作用,為后續(xù)研究提供了切入點(diǎn)。通過(guò)解剖觀察、Tunnel染色和AnnexinⅤ細(xì)胞凋亡流式檢測(cè)確定了成蜱吸血過(guò)程中細(xì)胞凋亡參與唾液腺的變化過(guò)程;為了探究半胱氨酸蛋白酶在過(guò)程中的作用機(jī)制,利用顯微注射技術(shù)將體外合成的干擾用CASP1雙鏈RNA注射入鐮形扇頭蜱成蜱體內(nèi),24小時(shí)后接種新西蘭大白兔,分別對(duì)上體后1到7天的對(duì)照組與實(shí)驗(yàn)組成蜱唾液腺進(jìn)行分離觀察,在mRNA和蛋白水平上檢測(cè)7天吸血過(guò)程中半胱氨酸蛋白酶的動(dòng)態(tài)變化,利用Tunnel細(xì)胞凋亡染色和AnnexinⅤ流式細(xì)胞凋亡檢測(cè)技術(shù)對(duì)吸血過(guò)程中唾液腺的變化進(jìn)行監(jiān)測(cè),結(jié)果表明進(jìn)入快速吸血期前(上體3~4天)是唾液腺細(xì)胞誘導(dǎo)細(xì)胞凋亡發(fā)生的主要時(shí)相,隨后隨著吸血過(guò)程快速完成,唾液腺細(xì)胞逐漸壞死,CASP1作為效應(yīng)性半胱天冬氨酸蛋白酶,在上體后5到7天表達(dá)量逐漸增加;CASP1的干擾可明顯延長(zhǎng)唾液腺細(xì)胞的凋亡,影響吸血過(guò)程中唾液腺細(xì)胞的壞死,并且誘導(dǎo)ATG4D的表達(dá)量逐漸增加,表明ATG4D與CASP1之間關(guān)系密切,暗示了自噬蛋白酶與半胱天冬氨酸蛋白酶之間可能存在互補(bǔ)的代償關(guān)系,共同參與唾液腺細(xì)胞的凋亡壞死過(guò)程。綜合上述結(jié)果,本研究鑒定了鐮形扇頭蜱6個(gè)半胱氨酸蛋白酶分子,發(fā)現(xiàn)它們?cè)隍缥笸僖合僦猩险{(diào)表達(dá),相互之間存在一定互作調(diào)節(jié)關(guān)系,其中自噬相關(guān)的半胱氨酸蛋白酶與凋亡相關(guān)的半胱氨酸蛋白酶共同參與了蜱唾液腺的凋亡過(guò)程。
[Abstract]:Cysteine proteases are ancient and conserved proteases, which are widely distributed in various tissues and organs from viruses, bacteria to mammals. They are involved in embryonic development, antigen presentation, apoptosis and homeostasis maintenance, and are important physiological activities. The salivary gland is the first contact organ for the blood of the host to enter the tick body, and it is also the channel through which the pathogen in the tick body transmits to the host body. Disease control is very important; with the completion of blood sucking, the salivary glands of ticks gradually atrophy, which is caused by apoptosis, but the molecular mechanism is unclear. Sequencing and differential analysis of salivary gland transcriptomes of Pseudostellaria falciformis before and after blood sucking showed that 1179 genes were up-regulated and 574 genes were down-regulated, of which 25 (13 up-regulated genes EST and 1 down-regulated gene EST) were predicted in the total transcription library. Six full-length sequences of cysteine proteases were obtained and named cathepsin B (CATB), cathepsin L (CATL), caspase-1 (CASP1), caspase-8 (CASP8), autophagy-associated protease 4B (ATG4B) and autophagy-related protease 4D (ATG4D) respectively according to their gene structure and bioinformatics analysis. The transcription levels of 6 cysteine proteases in different stages of development and different blood-sucking states of ticks were detected by PCR. The results showed that the transcription levels of all proteases in young and nymph ticks were significantly down-regulated, while in adult ticks were significantly up-regulated. The transcription levels of 6 proteases were up-regulated in salivary glands of adult ticks, especially CAT B and C. The expression and purification of five cysteine proteases (CASP8 was not expressed) were carried out in vitro, and specific mouse antisera were prepared respectively. The activity of the recombinant proteins showed that the proteins exhibited certain activity in moderate concentration and PH range. Sex: The optimum pH of CASP1 was 5.5, reaching 60% of the activity of the positive control enzyme. Cathepsin B and L were inhibited with the increase of the concentration. Cathepsin L contained an inhibitory domain of 60 amino acids, and the activity of cathepsin L was significantly increased after excision of the domain. ATG4B and ATG4D have certain activity to the specific substrate of CASP1, up to 40% of the positive control. ATG4D has good activity to the specific substrate of cathepsin, and also has inhibitory effect at high concentration. The serum of the two cathepsin CATB and CATL had two bands between 35 kDa and 40 kDa, and the size of the autophagy-related protease was ~36 kDa (ATG4B) and ~40 kDa (ATG4D), and the size of the CASP1 protein was about 37 kDa, similar to dimerization at about 70 kDa. Immunohistochemical localization of five cysteine proteases in salivary glands and intestinal cavity showed that five proteases were widely distributed in salivary glands, while only a small amount of ATG4D was found in the midgut. In nymph ticks of Pseudostellaria falciparum, a single interference test was conducted to detect the interaction between six cysteine proteases. The results showed that inhibition of any protease could cause changes in the transcriptional levels of the other five proteases, especially AT when the transcription of CAT B and CAT L was inhibited. The transcriptional levels of G4B and CASP1 were significantly up-regulated, while those of CASP8 and ATG4D were also significantly up-regulated. When CASP1 was inhibited, the transcriptional levels of CAT B were significantly down-regulated. The transcriptional levels of ATG4D, ATG4B and CASP8 were also significantly inhibited, but the transcriptional levels of CAT L were not affected. TG4D may play an upstream regulatory role, providing a starting point for further research. Through anatomical observation, Tunnel staining and Annexin V cell apoptosis flow cytometry, we determined that apoptosis participated in the process of salivary gland changes in adult ticks during blood sucking. Disruption of biosynthesis in vitro was injected into adult ticks of Pseudostellaria falciformis with CASP1 double-stranded RNA. New Zealand rabbits were inoculated with CASP1 double-stranded RNA 24 hours later. The salivary glands of the control group and the experimental group were separated and observed 1 to 7 days later. The dynamic changes of cysteine proteinase were detected at the level of mRNA and protein during 7 days of blood sucking. Cell apoptosis staining and Annexin V flow cytometry were used to monitor the changes of salivary glands during blood sucking. The results showed that the main phase of apoptosis induced by salivary gland cells was before the fast blood sucking stage (3-4 days from the upper body). With the rapid completion of the blood sucking process, salivary gland cells gradually necrosis, and CASP1 acted as an effect. The expression of caspase increased gradually from 5 to 7 days after supernatant. The interference of CASP1 could significantly prolong the apoptosis of salivary gland cells, affect the necrosis of salivary gland cells during blood sucking, and induce the expression of ATG4D to increase gradually, indicating that there was a close relationship between ATG4D and CASP1, suggesting that autophagy protease and caspase were closely related. There may be complementary compensatory relationship between proteases involved in the process of apoptosis and necrosis of salivary gland cells. Based on the above results, six cysteine proteinase molecules from ticks were identified. They were up-regulated in the salivary gland of ticks after blood sucking, and there was a certain interaction between them, among which autophagy was related. Cysteine protease and apoptosis-related cysteine protease participate in the apoptosis process of tick salivary gland.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.7
，
本文編號(hào)：2176986
[Abstract]:Cysteine proteases are ancient and conserved proteases, which are widely distributed in various tissues and organs from viruses, bacteria to mammals. They are involved in embryonic development, antigen presentation, apoptosis and homeostasis maintenance, and are important physiological activities. The salivary gland is the first contact organ for the blood of the host to enter the tick body, and it is also the channel through which the pathogen in the tick body transmits to the host body. Disease control is very important; with the completion of blood sucking, the salivary glands of ticks gradually atrophy, which is caused by apoptosis, but the molecular mechanism is unclear. Sequencing and differential analysis of salivary gland transcriptomes of Pseudostellaria falciformis before and after blood sucking showed that 1179 genes were up-regulated and 574 genes were down-regulated, of which 25 (13 up-regulated genes EST and 1 down-regulated gene EST) were predicted in the total transcription library. Six full-length sequences of cysteine proteases were obtained and named cathepsin B (CATB), cathepsin L (CATL), caspase-1 (CASP1), caspase-8 (CASP8), autophagy-associated protease 4B (ATG4B) and autophagy-related protease 4D (ATG4D) respectively according to their gene structure and bioinformatics analysis. The transcription levels of 6 cysteine proteases in different stages of development and different blood-sucking states of ticks were detected by PCR. The results showed that the transcription levels of all proteases in young and nymph ticks were significantly down-regulated, while in adult ticks were significantly up-regulated. The transcription levels of 6 proteases were up-regulated in salivary glands of adult ticks, especially CAT B and C. The expression and purification of five cysteine proteases (CASP8 was not expressed) were carried out in vitro, and specific mouse antisera were prepared respectively. The activity of the recombinant proteins showed that the proteins exhibited certain activity in moderate concentration and PH range. Sex: The optimum pH of CASP1 was 5.5, reaching 60% of the activity of the positive control enzyme. Cathepsin B and L were inhibited with the increase of the concentration. Cathepsin L contained an inhibitory domain of 60 amino acids, and the activity of cathepsin L was significantly increased after excision of the domain. ATG4B and ATG4D have certain activity to the specific substrate of CASP1, up to 40% of the positive control. ATG4D has good activity to the specific substrate of cathepsin, and also has inhibitory effect at high concentration. The serum of the two cathepsin CATB and CATL had two bands between 35 kDa and 40 kDa, and the size of the autophagy-related protease was ~36 kDa (ATG4B) and ~40 kDa (ATG4D), and the size of the CASP1 protein was about 37 kDa, similar to dimerization at about 70 kDa. Immunohistochemical localization of five cysteine proteases in salivary glands and intestinal cavity showed that five proteases were widely distributed in salivary glands, while only a small amount of ATG4D was found in the midgut. In nymph ticks of Pseudostellaria falciparum, a single interference test was conducted to detect the interaction between six cysteine proteases. The results showed that inhibition of any protease could cause changes in the transcriptional levels of the other five proteases, especially AT when the transcription of CAT B and CAT L was inhibited. The transcriptional levels of G4B and CASP1 were significantly up-regulated, while those of CASP8 and ATG4D were also significantly up-regulated. When CASP1 was inhibited, the transcriptional levels of CAT B were significantly down-regulated. The transcriptional levels of ATG4D, ATG4B and CASP8 were also significantly inhibited, but the transcriptional levels of CAT L were not affected. TG4D may play an upstream regulatory role, providing a starting point for further research. Through anatomical observation, Tunnel staining and Annexin V cell apoptosis flow cytometry, we determined that apoptosis participated in the process of salivary gland changes in adult ticks during blood sucking. Disruption of biosynthesis in vitro was injected into adult ticks of Pseudostellaria falciformis with CASP1 double-stranded RNA. New Zealand rabbits were inoculated with CASP1 double-stranded RNA 24 hours later. The salivary glands of the control group and the experimental group were separated and observed 1 to 7 days later. The dynamic changes of cysteine proteinase were detected at the level of mRNA and protein during 7 days of blood sucking. Cell apoptosis staining and Annexin V flow cytometry were used to monitor the changes of salivary glands during blood sucking. The results showed that the main phase of apoptosis induced by salivary gland cells was before the fast blood sucking stage (3-4 days from the upper body). With the rapid completion of the blood sucking process, salivary gland cells gradually necrosis, and CASP1 acted as an effect. The expression of caspase increased gradually from 5 to 7 days after supernatant. The interference of CASP1 could significantly prolong the apoptosis of salivary gland cells, affect the necrosis of salivary gland cells during blood sucking, and induce the expression of ATG4D to increase gradually, indicating that there was a close relationship between ATG4D and CASP1, suggesting that autophagy protease and caspase were closely related. There may be complementary compensatory relationship between proteases involved in the process of apoptosis and necrosis of salivary gland cells. Based on the above results, six cysteine proteinase molecules from ticks were identified. They were up-regulated in the salivary gland of ticks after blood sucking, and there was a certain interaction between them, among which autophagy was related. Cysteine protease and apoptosis-related cysteine protease participate in the apoptosis process of tick salivary gland.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.7
，
本文編號(hào)：2176986
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