流行性乙型腦炎病毒NS1蛋白單克隆抗體的制備及病毒特異性抗原

1、流行性乙型腦炎病毒NS1蛋白單克隆抗體的制備及病毒特異性抗原        【中文摘要】流行性乙型腦炎(Japanese encephalitis,JE)廣泛分布于東亞和南亞,是由乙型腦炎病毒(Japanese encephalitis virus,JEV)引起的嚴(yán)重的人畜共患傳染病。乙型腦炎病毒的NS1蛋白參與病毒的RNA復(fù)制,能誘發(fā)機(jī)體在非中和性抗體存在的情況下產(chǎn)生保護(hù)性免疫,但目前它們的機(jī)制尚不清楚。乙型腦炎病毒與西尼羅病毒(West Nile virus,WNV)、圣路易斯腦炎病毒(Saint Louis

2、encephalitis virus,SLEV)和墨累谷腦炎病毒(Murray Valley encephalitisvirus,MVEV)同屬一個血清群,這4種病毒有著極其相似的生物學(xué)特性,它們中的兩種或多種經(jīng)常在同一個地區(qū)流行,感染后導(dǎo)致相同或相似的臨床癥狀。目前的鑒別診斷方法主要包括噬斑減少中和試驗(plaque reduction neutralization test,PRNT)、病毒分離、RT-PCR等,這些方法不但費時費力,而且對實驗設(shè)備和職員具有一定要求,甚至需要生物安全3級實驗室,顯然這并不能滿足流行病學(xué)調(diào)查中大規(guī)樣子容貌品檢測和臨床上快速診斷的需要,因此需要建立一種具有高度

3、特異性的血清學(xué)診斷方法。本研究首先將乙型腦炎病毒疫苗株SA14-14-2株的NS1基因克隆到原核表達(dá)載體pET-30,重組質(zhì)粒驗證連接正確后轉(zhuǎn)化大腸桿菌感受態(tài)細(xì)胞BL21,經(jīng)IPTG誘導(dǎo)表達(dá)后,將表達(dá)產(chǎn)物純化作為免疫原,免疫6周齡BALB/c小鼠;經(jīng)過免疫、融合、篩選等過程,共獲得4株特異性針對NS1蛋白的單克隆抗體,IFA表明其中3株能識別自然狀態(tài)下的NS1蛋白。設(shè)計一套包含整個NS1蛋白的51個短肽片斷,每個肽段長度為16aa,每兩個相鄰的短肽之間有8aa的重疊。為表達(dá)這些短肽,首先人工合成了表達(dá)它們的基因,在編碼鏈的5'端引進(jìn)BamHI位點,3'端引進(jìn)XhoI位點。反義鏈

4、與編碼鏈互補(bǔ),且退火后形成的雙鏈DNA正向為BamHI粘性末端,反向為XhoI粘性末端,可直接與酶切線性化處理的pGEX-6P-1載體相連接;這51個肽段經(jīng)IPTG誘導(dǎo)后得到了表達(dá),表達(dá)產(chǎn)物與制備的單克隆抗體1H6作用,ELISA結(jié)果顯示其中的第18(NS1-18)和第19(NS1-19)與單抗1H6呈現(xiàn)明顯的陽性反應(yīng),而其他49個短肽為陰性反應(yīng),由此可以判定單抗1H6的抗原表位位于NS1-18和NS1-19的重疊區(qū)域(1819-0);將該區(qū)域逐一氨基酸截短表達(dá)后與單抗1H6反應(yīng),結(jié)果表明序列146EHRAW150為該表位的核心序列,Western blot表明該表位可被豬JEV陽性血清所識別

5、。在GenBank中選取39株基因全部測序的JEV,序列比對顯示該表位在不同毒株之間高度守舊,同源性為100%;選取西尼羅病毒(West Nile virus,WNV)、圣路易斯腦炎病毒(SaintLouis encephalitis virus,SLEV)、墨累谷腦炎病毒(Murray Valley encephalitis virus,MVEV)、登革熱病毒(DENV)、蜱傳染性腦炎病毒(Tick-borne encephalitis virus,TBEV)和黃熱病毒(Yellow Fever virus,YFV)共162株其他相關(guān)的黃病毒科病毒,將其NS1蛋白上的相關(guān)區(qū)域與該抗原表位進(jìn)行

6、序列比對,結(jié)果表明該抗原表位在不同黃病毒科病毒之間守舊性差;為確定所鑒定表位的特異性,將這162株病毒NS1蛋白上與該抗原表位同源區(qū)域表達(dá)后與單抗1H6反應(yīng),結(jié)果顯示所有其他病毒NS1蛋白的同源區(qū)域均不與單抗1H6反應(yīng),表明在本實驗中所鑒定的表位為病毒特異性抗原表位。本研究首次確定了位于乙型腦炎病毒NS1蛋白上的一個病毒特異性抗原表位,為建立一種方便、快捷、適用于現(xiàn)地大規(guī)樣子容貌品檢測的鑒別診斷方法奠定了基礎(chǔ),同時也為乙型腦炎病毒新型亞單位疫苗的研制,以及研究病毒感染和機(jī)體免疫過程中NS1蛋白與宿主體內(nèi)相應(yīng)分子之間的相互作用提供了有用信息。');【Abstract】 Japanese

7、encephalitis (JE), a serious zoonosis, is widely distributed in most of East and South-east Asia and partly in Oceania. JE is caused by Japanese encephalitis virus (JEV), the NS1 protein of JEV play roles in viral RNA replication and is able to induce protective immunity without neutralization antib

8、ody, but those definite mechanisms are less clear. Except JEV, the Japanese encephalitis virus serocomplex of the family Flaviviridae includes West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). These viruses have a similar ecology, it is very co

9、mmon that two or more of these flaviviruses co-circulate in some regions of the world, the initial symptoms of most of these viral infections are similar to each other. Although the plaque reduction neutralization test (PRNT), virus isolation and reverse transcription-polymerase chain reaction (RT-P

10、CR) are generally conducted to achieve laboratory diagnosis, these methods are time-consuming or involve manipulation of live virus which requires high level of biosafety laboratory, and not amenable to testing large numbers of specimen. Hence the development of a new serodiagnosis method is necessa

11、ry.In this study, the NS1 gene of JEV SA14-14-2 strain was cloned to pET-30 vector and transformed into Escherichia coli BL21 cells for expression. After IPTG induction, the fusion protein was purified and used to immunize BALB/c mice. After the procedure of immunization, cell fusion and selection,

12、4 strains of hybridoma were generated. IFA suggested that three of these mAbs recognized native NS1 protein. Then a library of 51 peptides spanning the entire NS1 protein was designed for the purpose of mapping epitopes broadly. Each peptide was 16 amino-acid in length and adjacent peptides had 8 am

13、ino-acid residues in common; To express these polypeptides, we synthesized complementary oligonucleotide pairs encoding each peptide, a BamHI site and XhoI site were added to the 5' and 3' ends of coding sequence, respectively. Then the complementary oligonucleotide pairs were annealed and c

14、loned into the BamH I and Xho I sites of pGEX-6p-1. The GST fusion proteins were expressed after IPTG induction and screened by indirect ELISA. Results showed that NS1-18 and NS1-19 were recognized by the mAbs 1H6 equally whereas others were not. So we deduce that the epitope of mAb 1H6 locates in t

15、he overlapping domain of NS1-18 and NS1-19. This region and 10 peptides with deletions were expressed to identify the epitope precisely. ELISA and Western blot indicated that 146EHRAW150 is the minimal unit of this epitope, and it can be recognized by positive serum from pigs.To investigate the homo

16、logy of the epitope among flavivirus, sequence alignment of the epitope and other peptides from the homologous region of NS1 protein of 39 JEV strains was completed. It was found that this linear epitope is totally conserved among these JEV strains. Then other 162 flavivirus strains, including the m

17、embers of JE serocomplex of WNV. SLEV and MVEV and another three antigenically related flavivirus, Dengue virus(DENV, type 14), Yellow Fever virus (YFV) and Tick-borne encephalitis virus (TBEV) were selected for alignment analysis. Alignment result showed that this epitope is less conserved among ot

18、her flavivirus strains. According to the alignment result, 12 clones represent the same domains as this JEV-NS1 epitope on other flavivirus NS1 protein were obtained and screened with Western bolt, it was found that all the homologous sequences of other 162 flavivirus strains were not reactive with mAb 1H6. The data obtain in current study indicated that this epitope is a JEV-specific epitope.This is the first time to identify a virus-specific epitope on NS1 protein of