凡纳滨对虾鳃细胞膜IHHNV衣壳蛋白受体的筛选

Preliminary screening for infectious hypodermal and hematopoietic necrosis viruscapsid protein receptors from Litopenaeus vannamei gill membrane proteins

对虾传染性皮下及造血组织坏死病(IHHN)为对虾主要病毒病之一,近些年,在全球范围内广泛流行并造成严重的经济损失。目前,在流行病学和诊断方面受到重视,但其致病机理鲜有报道。本研究首先构建了IHHNV CP原核表达载体,纯化CP蛋白并制备多抗,抗体效价达到1︰51200;利用匀浆、超速离心方法分离未感染IHHNV的凡纳滨对虾(Litopenaeus vannamei)鳃细胞膜蛋白;用VOPBA和HIS PULL–DOWN方法筛选凡纳滨对虾鳃细胞膜IHHNV CP受体,分别将疑似的蛋白条带进行LC-MS/MS分析。结果表明,信号转导与转录激活因子(STAT)、热休克蛋白90(HSP 90)、酚氧化酶原2型、Na+/K+-ATP酶α亚基4种蛋白能与IHHNV衣壳蛋白产生相互作用,并具有多种生物学活性,可能参与病毒的入侵及细胞病变的产生。其具体作用有待深入研究。

Infectious hypodermal and hematopoietic necrosis virus（IHHNV） is one of the most serious diseases of shrimp, and has a wide host range. IHHNV is the smallest of all known shrimp viruses and is a non-enveloped, linear, single-stranded DNA virus in Brevidensovirus Densovirinae Parvoviridae. This disease causes serious economic losses worldwide. IHHNV is highly pathogenic and is associated with high Litopenaeus stylirostrismortality rates. IHHNV causes chronic infection, slow growth, lesions in the forehead sword, antenna, and the cephalothoracic and abdominal shell of Litopenaeus vannamei. Since IHHNV was first identified, many studies have established diagnostic methods, and epidemiological surveys have been conducted, but the pathogenic mechanisms of IHHNV infection remain relatively unknown. In this study, we identified putative host cell receptors for the IHHNV capsid protein（CP） in L. vannamei gill membranes. DNA was extracted from IHHNV-infected L. vannamei（Sheyang isolate） and preserved at the Microbiology and Immunology, Preventive Veterinary Laboratory of Nanjing Agricultural University. The IHHNV CP gene was amplified by polymerase chain reaction, and the IHHNV CP BL21-p ET-28a-CP prokaryotic expression vector was prepared. The protein was expressed primarily in the precipitate. The CP in the precipitate was purified using a His Trap？ HP column and then used to prepare a polyclonal antibody. The antibody titer reached 1︰51200 after three immunizations. A Western blot analysis demonstrated that the polyclonal CP antibody bound specifically to CP. Then, gill membrane proteins from uninfected L. vannamei were obtained from a homogenate and ultracentrifuged to obtain a uniform gill membrane protein distribution. A virus overlay protein binding assay（VOPBA） and a His pull-downassaywere carried out to localize the IHHNV CP receptors. VOPBA is a classic method used to identify virus receptors and has been used to identify the host cell receptors for White Spot Syndrome Virus and Yellow Head Virus. The suspected protein strips were analyzed by mass spectrometry. Results of the two assays indicated that signal transducer and activator of transcription（STAT）, heat shock protein 90（HSP90）, prophenoloxidase-2, and the Na＋/K＋-ATPase alpha subunit participate in IHHNV infection. These four proteins have various biological functions, such as interferon signal transduction, protection against stress, defense, and maintenance of osmotic balance. These proteins may interact with the IHHNV CP and are associated with penetration and the cytopathic effects caused by IHHNV. Few studies have evaluated putative IHHNV CP receptors, probably because of the lack of a mature shrimp cell line. We used traditional VOPBA to localize the CP receptors from L. vannamei gill membrane proteins. Taken together, STAT, HSP90, prophenoloxidase-2, and the Na＋/K＋-ATPase alpha subunit are involved in IHHNV infection. These results will lay the foundation to identify the IHHNV invasive mechanism. The specific effects of the four proteins remain to be studied in detail.