Cloning, Serological Study And Monoclonal Antibodies Preparation Of S1 Domain In Spike Protein Of SARS-CoV

Severe acute respiratory syndrome (SARS) is a new emerging infectious disease. A new coronavirus, named SARS associated coronavirus (SARS-CoV), has been identified as the etiologic agent of SARS. Spike (S) glycoprotein, envelope (E), membrane (M), and nucleocapsid (N) proteins are the major four structure proteins of SARS-CoV, S, E and M proteins locate at the surface of virus particle but N protein at the core. S protein is the maximal structural protein and the predominant surface antigen, composing the characteristic tuber on the surface of SARS-CoV virus particle. The studies about previously identified coronaviruses and SARS-CoV both suggest S protein binds to the cell surface receptor and mediates subsequent fusion of the viral and cellular membranes, hence it is considered as the major target of antiviral drug development efforts as well as for developing vaccines and virus-neutralizing antibodies. S protein belongs to type I membrane glycoprotein, predicted to be 1255 amino acids in length, and has 24 potential N-linked glycosylation sites. It contains two distinct functional domains near the amino (S1) and carboxy (S2) termini, of the two parts, S1 forms the global domain and S2 forms the club-shaped domain crossing the membrane of SARS-CoV virus particle, and what is known that the peripheral S1 portion is responsible for binding to cellular receptors while the integral membrane S2 portion mediate the process of inducing membrane fusion for virus entry into target cells.Owing to a new coronavirus for the last SARS epidemic, it is still waiting for realizing roundly about the pathogenesis and biological character of SARS-CoV. Many studies have proved S protein plays a key role in the process of virus entry into target cells, and the virulence variation of SARS-CoV is close associated with themutation of SI domain of S protein. Therefore, cloning S protein gene, especially the SI domain, is critical for the viral pathogenesis study, seroepidemiological investigation and vaccine development for SARS-CoV, and producing the mAbs to the S protein of SARS-CoV may provide new material for the specific treatment of SARS and offer a basis to establish an antigen-capture sandwich ELISA detecting the S protein for the exploring of new early diagnostic method for SARS.This study aimed to clone the SI gene and the fragments derived of it into the prokaryotic expression vector for its expression, and produce the murine mAbs against S protein by immunize BALB/c mice using the purified immunogenic fusion protein and further establish a sandwich ELISA for detecting the S protein antigen based on the mAbs subquently to select the virus-neutralizing effect of mAbs to SARS-CoV and explore the value of antigen-capture ELISA for SARS early diagnosis, besides, set up a new serological diagnostic method for SARS based on the immunogenic fusion protein and compare it with another two serological diagnostic methods based on recombinant N protein and crude lysate of SARS-CoV respectively to explore the merits of the new diagnostic method for SARS serological diagnosis.The research has been divided into three parts and described as follows:I. Cloning, purification, and antigenic characterization of three recombinant fragments derived from SARS-CoV SI domainPreparing antigenic S protein would provide a means of study function of S protein, and production of mAbs against S protein for the diagnosis, and treatment SARS. This part of the study aimed to clone SI domain gene and the fragments derived from SI into the prokaryotic expression vector and provide an excellent immnogen for the production of the mAbs against S protein and the establishment of serological diagnostic method for SARS in the following studies. Firstly, the SI domain gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pQE-30. Three fragments (40~751, 746~1 344, and 746~2 001 bp) derived from SI domain produced after the recombinant plasmid pQE-30/Sl was digested by restriction endonucleases respectively and subsequently all were cloned into pQE-30 and expressed in M15 strains of Escherichia coli. The expression products, 26 700 dalton, 22 500 dalton, and 46 000 dalton fusion proteins, designated SI a, Sib, and Sic respectively, were purified successfully with Ni affinity chromatography. The immunogenicity of the three recombinant proteins was analyzed by Western Blot and ELISA using serologically confirmed sera from SARS patients. A recombinant Sic protein derived from S protein (249~667 aa, including the receptor binding domain) could react to specific antibodies in SARS patients' sera.These results suggested that Sic protein possesses excellent immunogenicity.n. Production and characterization of monoclonal antibodies against Sic derived from SI domain of SARS-CoVThere was no specific and effective treatment of SARS. Therefore, the murine virus-neutralizing mAbs might be the candidates of the biological medicaments for SARS-CoV. In addition, the antigen-capture ELISA based on the mAbs against S protein might be a new early diagnostic method for SARS. Based on the immunogenic recombinant Sic protein obtained in the first part of this study, this part aimed to prepare the mAbs against Sic protein to offer candidates of virus-neutralizing mAbs and establish an antigen-capture ELISA to detect the S protein. Firstly, the 6xHis-tagged recombinant fragment at N-terminal residues 249 to 667 of SI domain in SARS-CoV S protein, Sic fusion protein, was used to immunize the BALB/c mice for the mAbs preparation. The immunized splenocytes were fused with the NS-1 myeloma cells. After the hybridoma cells were subcloned with limited dilution, for the first time, three hybridomas producing mAbs steadily had been produced. Two of the mAbs were IgG2a isotype, and the other was IgGl. IF A and Western blotting characterized the high specificity of the mAbs with the Vero cells infected with SARS-CoV and none of the 3 mAbs were reactive with human coronaviruses 229E and OC43. Good affinity test results obtained when the affinity of the mAbs against SI domain was tested by indirect ELISA, the affinity constant of the 3 mAbs were 9.96xlO~8, 1.62xlO~10, and 8.45xlO"9, respectively. Competitive inhibition assay showed that the two mAbs recognized at least two distinct epitopes of S antigen. To establish optimal antigen capture ELISA, capturing and detecting antibodies was selected by paring one mAb and two other mAbs conjugated HRP one by one. A high sensitive double mAbs based sandwich ELISA was established. The lowest limit of detection of the recombinant S protein with this capture ELISA is 3ng/ml approximately.HI. Study on the specific antibody against the Sic domain of S glycoprotein in patients with SARSAt present, the diagnosis reagent for SARS serological analysis was based on the crude lysate of SARS-CoV, low sensitivity, low specificity and high safe-level of its preparation were the major demerits. Evaluation of the serological diagnostic methods based on the recombinant structural proteins of SARS-CoV for SARS serological diagnosis probably win a feasible substituted serological diagnostic method for that of based on crude lysate of SARS-CoV. This part of the study aimed to set up a new indirect ELISA for the serological analysis of SARS based on thepurified recombinant Sic protein to understand the humoral immunity to the Sic protein of SARS-CoV and the possibility of using Sic protein in SARS diagnosis. Antibody IgG to the Sic protein was analyzed from 86 patients who had SARS, as diagnosed on the basis of World Health Organization criteria. The results of the Sic protein ELISA were compared with results from a commercially available ELISA kit based on lysates of SARS-CoV as antigen and N protein ELISA. The results suggested the positive rate for the specific antibody IgG against Sic protein increased with time in the course of disease, in accordance with those of the specific antibodes IgG against SARS-CoV and N protein. The positive rates of the anti-Slc IgG within the first week after onset of disease were 5% (2/44) and increased up to 39% (7/18) for the second week. 83% (20/24) of the serum specimens were positive for the anti-Slc IgG collected 15 or more days after illness. Comparatively, the positive rates of the anti-N IgG, just as that of SARS-CoV IgG, were 14% (6/44), 56% (10/18) and 100% (24/24) in the three phases, respectively. The result of the ELISA assay for Sic showed 83% (76/86) and 80% (71/86) agreements with those of the indirect ELISA based on the crude lysate of SARS-CoV and recombinant N protein respectively. In the serum specimens from health donors, no positive result obtained for the anti-Slc IgG detection, which suggested Sic protein possesses excellent specificity, but the specific anti-N protein IgG was detected in a few samples (1.88%, 14/745) by Western Blot and ELISA. In conclusion,1. The subsection genes derived from SI domain of S protein were cloned into prokaryotic expression vector pQE-30, subsequently expressed and purified successfully. For the first time, one recombinant protein SI was obtained which including the epitopes recognized by the virus-neutralizing antibodies and possessing excellent immunogenicity. This study provided a good immunogen for the virus-neutralizing mAbs preparation and the establishment of a new serological diagnostic method for SARS.2. Three mAbs specific against SI domain of S protein obtained for the first time. And based on the 3 mAbs, a new sandwich ELISA was established to detect the S protein for SARS diagnosis. This study provided candidates of virus-neutralizing mAb and a new possible tool for the early diagnosis of SARS.3. The serological study on recombinant Sic protein showed that the specific IgG against Sic was detected in most of the serum specimens from convalescent patients with SARS. This result indicates that the ELISA based on the SARS-CoV S protein has high sensitivity. The recombinant S protein of SARS-CoV may be a gooddiagnostic marker for serodiagnosis of SARS.