殘緣璃眼蜱subolesin和半胱氨酸蛋白酶基因生物學(xué)功能初步研究
發(fā)布時(shí)間：2018-07-28 18:02
【摘要】：殘緣璃眼蜱作為專性吸血外寄生蟲,能夠傳播多種病原體(特別是環(huán)形泰勒蟲),對(duì)畜牧業(yè)造成巨大的經(jīng)濟(jì)損失。目前對(duì)于蜱以及蜱傳病的防治方法主要以化學(xué)藥物治療為主,但由此引發(fā)的環(huán)境污染、蜱的抗藥性等問題越來越嚴(yán)重,因此免疫學(xué)防治方法逐漸被重視起來。免疫學(xué)方法重要的基礎(chǔ)是尋找合適的蜱的功能基因作為保護(hù)性抗原,為抗蜱及蜱傳病疫苗提供理論性基礎(chǔ)。根據(jù)已有的蜱種功能基因的研究表明,subolesin和半胱氨酸蛋白酶基因存在于多種生物體中;subolesin基因在高等真核生物中具有較高的同源性,在蜱種中具有較好的保守性;半胱氨酸蛋白酶(CP)與宿主血液消化及蜱的生長發(fā)育相關(guān)。試驗(yàn)首次從殘緣璃眼蜱、亞洲璃眼蜱中擴(kuò)增出subolesin基因ORF,大小均為492 bp,編碼163個(gè)氨基酸。生物信息學(xué)分析表明,subolesin基因在各蜱種間具有高度保守性,且有一定的種間特異性和屬間差異性。將subolesin基因連接到pGEX-4T-1載體上進(jìn)行重組表達(dá),重組蛋白大小約為45 ku。通過SDS-PAGE和Western blotting分析顯示,體外表達(dá)重組蛋白以包涵體形式存在,純化的目的蛋白與兔抗殘緣璃眼蜱饑餓成蜱血清、抗蜱唾液腺和抗蜱卵巢血清發(fā)生免疫反應(yīng)。實(shí)時(shí)熒光定量分析表明,subolesin基因存在于蜱的各個(gè)發(fā)育階段和組織,但相對(duì)表達(dá)豐度不同,中腸、唾液腺的表達(dá)豐度相對(duì)較高,在蜱吸血前后表達(dá)豐度有很大差異,成蜱吸血過程中subolesin基因表達(dá)豐度出現(xiàn)先上調(diào)后下調(diào)的現(xiàn)象;動(dòng)物保護(hù)試驗(yàn)和RNAi試驗(yàn)證明,蜱的吸血率、飽血體重、飽血時(shí)間、產(chǎn)卵率、卵的孵化率都受到影響;這些現(xiàn)象表明該基因可能與蜱的生長、發(fā)育及吸血相關(guān)。本試驗(yàn)利用通用引物cys500首次從殘緣璃眼蜱中獲得CP保守序列,通過5'RACE和3'RACE擴(kuò)增技術(shù)獲得CP基因全長。CP基因全長為1192 bp,包含一個(gè)999 bp的ORF。生物學(xué)軟件分析CP基因在不同蜱種間具有一定的保守性。連接到pET30a載體上成功表達(dá),重組蛋白約為45 ku;SDS-PAGE和Western blotting分析顯示,體外表達(dá)的融合蛋白以包涵體的形式存在,并和兔抗殘緣璃眼蜱饑餓成蜱血清發(fā)生免疫反應(yīng),表明該融合蛋白具有良好的反應(yīng)原性。實(shí)時(shí)熒光定量分析顯示,CP基因表達(dá)具有選擇性,組織中僅中腸有較高的表達(dá)豐度,其他組織中相對(duì)表達(dá)豐度較低,基本可以忽略,蜱卵中該基因相對(duì)表達(dá)豐度較高,蜱吸血過程中基因表達(dá)豐度出現(xiàn)上調(diào)現(xiàn)象;動(dòng)物保護(hù)性試驗(yàn)和RNAi結(jié)果表明:蜱的吸血、飽血程度及產(chǎn)卵情況受到影響,證明CP可能與蜱的生長、發(fā)育及卵發(fā)育相關(guān)。
[Abstract]:As a specific blood sucking parasite, ticks can transmit a variety of pathogens (especially Taylor ringworm), causing great economic losses to animal husbandry. At present, the main control methods for ticks and tick-borne diseases are chemotherapeutic treatment. However, the environmental pollution and the resistance of ticks are becoming more and more serious, so immunological methods have been paid more and more attention. The important basis of immunological methods is to find suitable functional genes of ticks as protective antigens and to provide theoretical basis for anti-tick and tick-borne vaccines. According to the existing functional genes of ticks, subolesin and cysteine protease genes were found to have high homology in higher eukaryotes and good conserved in ticks. Cysteine protease (CP) is associated with host blood digestion and the growth and development of ticks. For the first time, the subolesin gene ORF was amplified from ticks residualis and ticks asiatica, with a size of 492bp, encoding 163 amino acids. Bioinformatics analysis showed that the subolesin gene was highly conserved among the ticks, and had some interspecific and intergeneric differences. The subolesin gene was ligated into the pGEX-4T-1 vector for recombinant expression, and the size of the recombinant protein was about 45 ku. SDS-PAGE and Western blotting analysis showed that the recombinant protein expressed in vitro existed in the form of inclusion body. The purified target protein reacted with the sera of adult ticks, anti-tick salivary glands and anti-tick ovaries in rabbits. Real-time fluorescence quantitative analysis showed that the subolesin gene existed in various stages and tissues of ticks, but the relative expression abundance was different, and the expression abundance of midgut and salivary gland was relatively high. The expression abundance of subolesin gene was up-regulated and then down-regulated in adult ticks, the animal protection test and RNAi test showed that the rate of blood absorption, body weight, time, oviposition rate and hatching rate of ticks were all affected. These phenomena suggest that the gene may be related to the growth, development and blood absorption of ticks. The CP conserved sequence was obtained by using universal primer cys500 for the first time. The total length of CP gene was 1192 BP, including a 999bp ORF by 5'RACE and 3'RACE amplification. The analysis of CP gene by biological software was conserved among different ticks. The fusion protein was expressed in the form of inclusion body and immunoreactive with adult ticks sera from rabbits. The recombinant protein was about 45ku-SDS-PAGE and Western blotting analysis showed that the fusion protein expressed in vitro existed in the form of inclusion body. The results showed that the fusion protein had good reactivity. The results of real-time fluorescence quantitative analysis showed that the expression of CP gene was selective, only in the midgut, but in other tissues was relatively low, which could be neglected, and the relative expression abundance of the gene in ticks eggs was higher than that in other tissues. The results of animal protective test and RNAi showed that the degree of blood intake and oviposition of ticks were affected, which suggested that CP might be related to the growth, development and egg development of ticks.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
[Abstract]:As a specific blood sucking parasite, ticks can transmit a variety of pathogens (especially Taylor ringworm), causing great economic losses to animal husbandry. At present, the main control methods for ticks and tick-borne diseases are chemotherapeutic treatment. However, the environmental pollution and the resistance of ticks are becoming more and more serious, so immunological methods have been paid more and more attention. The important basis of immunological methods is to find suitable functional genes of ticks as protective antigens and to provide theoretical basis for anti-tick and tick-borne vaccines. According to the existing functional genes of ticks, subolesin and cysteine protease genes were found to have high homology in higher eukaryotes and good conserved in ticks. Cysteine protease (CP) is associated with host blood digestion and the growth and development of ticks. For the first time, the subolesin gene ORF was amplified from ticks residualis and ticks asiatica, with a size of 492bp, encoding 163 amino acids. Bioinformatics analysis showed that the subolesin gene was highly conserved among the ticks, and had some interspecific and intergeneric differences. The subolesin gene was ligated into the pGEX-4T-1 vector for recombinant expression, and the size of the recombinant protein was about 45 ku. SDS-PAGE and Western blotting analysis showed that the recombinant protein expressed in vitro existed in the form of inclusion body. The purified target protein reacted with the sera of adult ticks, anti-tick salivary glands and anti-tick ovaries in rabbits. Real-time fluorescence quantitative analysis showed that the subolesin gene existed in various stages and tissues of ticks, but the relative expression abundance was different, and the expression abundance of midgut and salivary gland was relatively high. The expression abundance of subolesin gene was up-regulated and then down-regulated in adult ticks, the animal protection test and RNAi test showed that the rate of blood absorption, body weight, time, oviposition rate and hatching rate of ticks were all affected. These phenomena suggest that the gene may be related to the growth, development and blood absorption of ticks. The CP conserved sequence was obtained by using universal primer cys500 for the first time. The total length of CP gene was 1192 BP, including a 999bp ORF by 5'RACE and 3'RACE amplification. The analysis of CP gene by biological software was conserved among different ticks. The fusion protein was expressed in the form of inclusion body and immunoreactive with adult ticks sera from rabbits. The recombinant protein was about 45ku-SDS-PAGE and Western blotting analysis showed that the fusion protein expressed in vitro existed in the form of inclusion body. The results showed that the fusion protein had good reactivity. The results of real-time fluorescence quantitative analysis showed that the expression of CP gene was selective, only in the midgut, but in other tissues was relatively low, which could be neglected, and the relative expression abundance of the gene in ticks eggs was higher than that in other tissues. The results of animal protective test and RNAi showed that the degree of blood intake and oviposition of ticks were affected, which suggested that CP might be related to the growth, development and egg development of ticks.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 呂文順,張平成,魏寶瑛,黃守云,俞生榮,蒲正w，
本文編號(hào)：2151161
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