Cloning and Sequence Analysis of the <i>MUC</i>-1 Gene of <i>Toxocara canis</i>

Ai-yun JIANG; Fang LI; Shao-ji CHEN; Rong-qiong ZHOU

doi:10.13718/j.cnki.xdzk.2020.03.011

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To study the molecular characteristics of the muc-1 gene in Toxocara canis, the full-length coding sequencing of Tc-muc-1 was amplified using PCR amplification, multiple sequence alignment (MSA) was made and a phylogenetic tree was constructed. The results showed that the entire sequence of Tc-MUC-1 gene was 531 bp in length, encoding a protein of 176 amino acids. In functional domains analysis, Tc-MUC-1 was found to contain one mucin domain consisting of 11 STSSSSA repeats and two ShKT domains. Gene ontology (GO) annotations showed that Tc-MUC-1 had the molecular function of protein binding and metal ion binding. Multiple sequence alignments revealed that Tc-MUC-1 and the other four mucins (Tc-MUC-2 to Tc-MUC-5) of T. canis all contained two ShKT domains composed of 36 amino acids. Phylogenetic analysis indicated that Tc-MUC-1 shared the highest levels of amino-acid sequence similarity with Pristionchus pacificus (Pristionchus pacificus; No. PDM76930) and Caenorhabditis elegans (Caenorhabditis elegans; No. NP491509). In conclusion, this study lays a foundation for further functional researches on Tc-MUC-1.

Corresponding author: Rong-qiong ZHOU

Received Date: 13/06/2019

Available Online: 20/03/2020

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Abstract: To study the molecular characteristics of the muc-1 gene in Toxocara canis, the full-length coding sequencing of Tc-muc-1 was amplified using PCR amplification, multiple sequence alignment (MSA) was made and a phylogenetic tree was constructed. The results showed that the entire sequence of Tc-MUC-1 gene was 531 bp in length, encoding a protein of 176 amino acids. In functional domains analysis, Tc-MUC-1 was found to contain one mucin domain consisting of 11 STSSSSA repeats and two ShKT domains. Gene ontology (GO) annotations showed that Tc-MUC-1 had the molecular function of protein binding and metal ion binding. Multiple sequence alignments revealed that Tc-MUC-1 and the other four mucins (Tc-MUC-2 to Tc-MUC-5) of T. canis all contained two ShKT domains composed of 36 amino acids. Phylogenetic analysis indicated that Tc-MUC-1 shared the highest levels of amino-acid sequence similarity with Pristionchus pacificus (Pristionchus pacificus; No. PDM76930) and Caenorhabditis elegans (Caenorhabditis elegans; No. NP491509). In conclusion, this study lays a foundation for further functional researches on Tc-MUC-1.

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弓首蛔虫病主要是弓首科(Toxocaridae)弓首属(Toxocara)犬弓首蛔虫(T. canis)寄生于人体及多种动物引起的一种重要的人兽共患病^[1].动物通过摄入其感染性虫卵或幼虫而感染.在终末宿主体内，T. canis感染性幼虫经内脏移行最终在肠道发育为成虫.在人及其他非特异宿主体内，感染性幼虫不能发育为成虫，而是移行到宿主诸多器官，如肝、肺、心、肾、眼睛、脑、肌肉等部位，成为滞育的第3期幼虫.当人体感染后，幼虫在移行期能够逃避宿主的免疫监视，因而不会产生明显的病理变化；而濒死和死亡的虫体则会诱发机体产生明显的病理免疫反应，引起内脏幼虫移行症(visceral larva migrans，VLM)、眼睛幼虫移行症(ocular larvemigrans，OLM)、神经弓首蛔虫病(neurological toxocariasis，NT)和隐性弓首蛔虫病(covert toxocariasis，CT)等，造成严重的病理综合征^[2-3].

黏蛋白(mucins，MUC)是一类由黏多糖组成的高分子量糖蛋白，主要存在于上皮组织中，具有PTS结构域，即含有丰富的丝氨酸(serine，P)、苏氨酸(threonine，T)和脯氨酸(proline，S)残基，这些残基都是高度氧糖基化，N端和C端富含天冬酰胺和半胱氨酸.黏蛋白在细胞黏附、分化、免疫应答、肿瘤形成以及细胞信号传导过程中具有多样化的生物学功能^[4].黏蛋白可分为分泌型黏蛋白和膜结合黏蛋白，分泌型黏蛋白(如MUC-2，MUC-5AC，MUC-5B)被包装成分泌物进入囊泡参与胞内信号转导；膜结合黏蛋白的羧基末端具有一个跨膜结构域，能将自身连接到质膜(Plasma membrane)(如MUC-1，MUC-3，MUC-4，MUC-16，MUC-17)，促进细胞—细胞/蛋白质—蛋白质结合，以及信号转导和蛋白质稳定^[5].

在哺乳动物组织中，黏蛋白为上皮表面提供黏性和物理保护，并携带大量对细胞运输和黏附起作用的聚糖^[6].黏蛋白1(MUC-1)是黏蛋白家族的重要成员，起着识别细胞、保护细胞以及维持黏性等作用^[7]. Tc-MUC-1是犬弓首蛔虫感染性幼虫分泌的一系列糖蛋白中的一种，参与机体免疫，对宿主免疫逃避发挥重要作用^[8].目前，国内外对Tc-MUC-1研究较少，本研究拟对犬弓首蛔虫黏蛋白1全长基因(Tc-muc-1)进行克隆及序列分析，以期为Tc-MUC-1的功能研究奠定基础.

1. 材料与方法

1.1. 材料

T.canis采自西南大学荣昌校区动物医院患病犬，经形态学鉴定后，保存于液氮罐中.

Taq DNA聚合酶、pMD19-T(simple)Vector、PrimeScript^TM反转录试剂盒购自TaKaRa公司；EasyPure©胶回收试剂盒、DH5α购自TransGen Biotech公司；Trizol试剂购自Ambion公司.

1.2. 方法

1.2.1. 引物的设计与合成

根据T.canis基因组数据(GenBank：U39815)，用PrimerPremier(Version5.0)软件设计引物，引物序列信息为上游引物：5'-ATGCACGTCCTTACCGT-3'；下游引物：5'-TTAACAGAAGCCGCACGT-3'.引物序列由重庆擎科兴业生物技术有限公司合成.

1.2.2. 总RNA的提取与反转录

将成熟的T.canis雌虫卵巢解剖，收集虫卵，在37 ℃培养箱中培养3~4周，采用Trizol试剂提取培养好的2期幼虫的总RNA^[9-10].采用核酸蛋白检测仪检测总RNA的浓度和纯度.以提取的T. canis幼虫总RNA为模板，按反转录试剂盒说明合成cDNA.

1.2.3. Tc-MUC-1基因的PCR扩增

以合成的T. canis幼虫cDNA为模板进行PCR扩增.扩增体系为25 μL：10×PCR buffer(不含Mg²⁺)2.0 μL、Mg²⁺(25 mmol/L)2.5 μL、dNTPs(2.5 mmol/L)2.0 μL、上游引物(10 μmol/L)0.5 μL、下游引物(10 μmol/L)0.5 μL、cDNA 1.0 μL、rTaq酶0.5 μL、dd H₂O 16.0 μL，以鸡异刺线虫cDNA为模板设置阴性对照. PCR反应条件为94 ℃预变性3 min，94 ℃变性30 s，55 ℃复性30 s，72 ℃延伸1 min，35个循环；最后72 ℃延伸5 min.扩增产物经1%琼脂糖凝胶电泳检测，并按DNA胶回收试剂盒说明，回收目的PCR产物.

1.2.4. Tc-MUC-1基因的克隆

将回收的目的PCR产物与pMD19-T(simple)Vector载体连接，将上述连接产物转化至DH5α感受态细胞，涂布于含Amp⁺(100 mg/mL)，X-Gal(20 mg/mL)和IPTG(24 mg/mL)的LB琼脂平板中，置恒温箱37 ℃培养12~14 h.

1.2.5. 测序及序列分析

将经菌液PCR鉴定为阳性的重组菌液送重庆擎科兴业生物技术有限公司测序.将测得的序列利用DNAStar 5.0等软件，分别从核酸和氨基酸水平对该基因进行生物学分析.利用SMART和InterProScan(http://smart.embl-heidelberg.de/)预测功能结构域.应用MUSCLE，Clustal Omega和MAFFT软件进行多重序列比对，用MEGA 5.0软件中的邻接法(Neighbour-joining，NJ法)构建系统进化树，进化树的可靠性用Bootstrap进行分析，共1 000个重复.

3. 讨论

黏蛋白(MUC)是高分子量的O-糖蛋白，主要表达于上皮细胞的顶端表面^[12].该蛋白在人乳中首次鉴定出来，几乎分布于所有呼吸腺体的上皮细胞表面、女性生殖道、胃肠道、中耳、唾液腺、乳腺、正常的胰脏内小叶导管等^[4]. MUC具有保护和润滑上皮细胞、维持上皮细胞特征、细胞黏附、分化和免疫的基本功能^[12-14].在肿瘤发生和其他病理状态下，MUC的表达发生显著变化^[15-16]，并可作为肿瘤诊断及(或)预后标志物.

本研究发现Tc-MUC-1结构包含黏蛋白结构域(Mucin domain)和ShKT结构域(ShKT domain)两部分.该蛋白序列富含丝氨酸残基，编码176个氨基酸且具有信号肽，但无跨膜结构域.多重序列比对显示Tc-MUC-1，Tc-MUC-2，Tc-MUC-3，Tc-MUC-4，Tc-MUC-5黏蛋白结构域的重复区域中Ser/Thr存在一定差异，即Tc-MUC-1富含Ser，而其余的黏蛋白具有不同的重复单位Thr，Gly，Pro和Ala，均具有Thr的优势.富含半胱氨酸的ShKT结构域具有高度的保守性，Tc-MUC-1，Tc-MUC-2，Tc-MUC-4的ShKT结构域由86%~97%相同的氨基酸残基构成. Loukas等^[6]发现富含半胱氨酸残基的ShKT结构域能促进黏蛋白三级结构的聚合和形成，表明该结构域在决定黏蛋白的功能中起重要作用.

MUC-1是一种膜结合蛋白，主要存在于上皮组织，是上皮组织黏液屏障的重要成分^[17]，已证明在人类多种器官(乳腺、肺、结肠、胃、前列腺)的恶性肿瘤中异常表达^[18-22]，参与多种恶性肿瘤类型的转移和侵袭. Gillh等^[23]发现秀丽隐杆线虫黏蛋白LET-653在排泄管和孔细胞的发育中起作用，并维持单细胞管型的完整性；Noya等^[24]发现肝片吸虫有一种黏蛋白样多肽，它与脂多糖协同促进树突状细胞成熟，并增强树突状细胞Th1极化能力，参与免疫调节作用. Leon-Coria等^[25]发现宿主胃肠道分泌的MUC-2在单层上皮细胞和腔内容物之间形成屏障，在先天防御溶组织内阿米巴(Entamoeba histolytica，Eh)和促炎反应中起重要作用.而Tc-MUC-1是否参与宿主细胞黏附、免疫识别及逃避、机体屏障等生物学过程，有待后续试验进一步研究.

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Cloning and Sequence Analysis of the MUC-1 Gene of Toxocara canis

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Cloning and Sequence Analysis of the MUC-1 Gene of Toxocara canis

Corresponding author: Rong-qiong ZHOU