Study Of SARS-CoV Spike Protein Gene Fragments DNA Vaccine And Its Immune Effects

Objects:The severe acute respiratory syndrome (SARS),also named infectious atypical pneumonia, is a emerging andhighly contagious respiratory infection that first occurred in late2002 in Guangdong Province, China, and spread to more than30 countries in early 2003,and lasted for about 7 months, closeto 8100 people were infected worldwide, among which 774people died ,With a mortality rate of over 9%.It has been identified that the pathogen of SARS is a novelcoronavirus, named as SARS-CoV. Absolutely, SARS-CoV hada major health and socioeconomic impact. Fortunately, therehave been very few incidences of SARS infections after the firstprevalence. However, with multiple modes of virus transmissionand a wide range of potential nonhuman reservoirs includingsome wild animals, it is highly likely that this disease will recurin the future. Currently, there are no effective antiviral drugs orvaccines available against this virus. To better control or preventfuture epidemics, some new anti-SARS-CoV vaccines need tobe developed.SARS-CoV is a 30 kb ssRNA positive-strand virus,belongs to Coronaviridae family. Similar to other knowncoronaviruses, the viral RNA genome has five major openreading frames (ORFs) and additional nine potential ORFs.These ORFs-encoded proteins include the replicase polyprotein,the spike (S), envelope (E), and membrane (M) glycoproteinsand the nucleocapsid protein (N). Among these proteins, spike(S)-glycoprotein of the virus interacts with a cellular receptorand mediates membrane fusion to allow viral entry intosusceptible target cells. Accordingly, S-protein plays animportant role in virus infection and is the primary target ofneutralizing antibodies.Considering DNA vaccination maybe a promising methodand S protein play a important role in immune responsesaccording to SARS, we chose two frgments from S gene as aimgene to construct the plasmid DNA vaccines and detect its'humoral and cell-mediated immune responses in immunizedmice, including one fragment containing the receptor-bindingdomain, which we thought are the most potential antigencandidates for vaccine design. We hope our study can explorethe possibility of DNA vaccine acting as SARS vaccine, laysome foundations for deeper understanding the immunology ofSARS-CoV and developing SARS vaccines.Methods:In the study, two gene fragments of the truncatedS protein of SARS-CoV named S1C and RBD were amplifiedby PCR with specific primers. One DNA truncated fragmentencoded 1609–2394bp of S gene(S1C)and the other encoded805-1614bp of S(RBD), which contained the target cellreceptor-binding domain. The amplified fragments were insertedinto pMD18-T vector to construct of the plasmid pMD18-S1Cand pMD18-RBD. Collected the fragments digested byrestrictive enzyme and subcloned them into eukaryotic vectorpVAX1 to construct eukaryotic plasmids pVAX-RBD andpVAX-S1C. Then transformed the two expression plasmids intoE coli XL1-Blue .The positive colonies were chosen andcultured overnight in LB medium containing kanamycin. Therecombinant plasmids were identified by PCR, double enzymedigestion and sequenced. Thus, the eukaryotic expressionplasmids pVAX-S1C and pVAX-RBD, containing the truncatedS gene of SARS-CoV was obtained. Transfected them into VeroE6 cells and 293T cells respectively in vitro to observe theirexpression in eukaryotic cells; Extracted and purified the twokinds of recombinant plasmids aboundantly. Then some BALB/cmice were randomly divided into different groups (five mice ineach group) and intramuscularly immuned with the recombinantplasmids pVAX-S1C or pVAX-RBD respectively. The mice inthe experimental group were injected with 200 mg plasmidsdissolved in 100ml PBS. And these mice were immunized moretwice with the same dosage at 2 weeks intervals. The mice incontrol group received the same amount of pVAX1 vector withidentical route and frequency. Collected the mice sera so tomeasure IgG antibody against SARS-CoV by enzyme-linkedimmunosorbent assay (ELISA) with E. coli expressed truncatedS protein RBD or SARS-CoV lysate. Moreover, microserumneutralizing test was performed significantly. Also, obtainingspleen cells of immunized mice, the specific cell-mediatedimmune responses were detected by flow cytometry (FCM) forthe ratio of T cell subgroup and enzyme-linked immunospot(ELISPOT) for secretion of γ-INF after specific stimulation.Results:(1) Two DNA fragments RBD and S1C both about800 bp were amplified by PCR using the plasmid containing thefull-length of spike gene as the template and two pair ofdesigned specific primers. (2) The two constructed plasmidspVAX-RBD and pVAX-S1C have been identified by PCR andenzyme digestion. Two electrophoretic bands about 800 bpcould be observed respectively under ultraviolet light after 1%agarase gel electrophoresis. The inserted fragments inrecombinant plasmids pVAX-RBD,pVAX-S1C were sequencedand compared their homology with SARS-Cov S gene of strainBJ01 from the Genbank: Both the sequences were totally same.(3) The plasmids pVAX-RBD could express protein RBD inVero E6 cells and pVAX-S1C express protein S1C in 293T cellssuccessfully. (4) Used the two plasmids pVAX-RBD andpVAX-S1C as DNA vaccines to immunize BALB/c mice. Thespecific IgG antibody against SARS-CoV could be measured byELISA with E. coli expressed truncated S protein RBD orSARS-CoV lysate as diagnostic antigen. The microserumneutralization assay showed the neutralizing antibodies wereappeared in the sera of all the five mice immunized withplasmid pVAX-RBD: The geomean antibody titer is 1:183.8.While the sera of the group with pVAX-S1C immunized and thecontrol group didn't show any neutralizing activity. Accordingto flow cytometry (FCM) analyzing, either group's ratio ofCD4+T cell of mice spleen cells with pVAX-S1C orpVAX-RBD immunized is higher than that of control group. (p<0.01) And enzyme-linked immunospot (ELISPOT) assayindicated the amount of spleen cell secreted IFN-γspecificallyof either recombinant plasmid immunized groups was alsohigher than that of control group immunized with pVAX1 vector.Statistics significance existed between pVAX-RBD and controlgroups. (p<0.05)Conclusion: (1) In this study, we choose SARS-CoV spikegene 805~1614 bp and 1609~2394 bp which contained thetarget cell receptor-binding domain as aim gene. The eukaryoticexpression plasmids pVAX-S1C and pVAX-RBD weresuccessfully constructed. (2) Plasmids pVAX-RBD andpVAX-S1C expressed in mammal cells with secreting formcould be detected by Western Blot or ELISA. It could be inferthat the two plasmids can express in eukaryotic cellssuccessfully. (3) Used the two plasmids pVAX-RBD andpVAX-S1C as DNA vaccines immunized BALB/c mice, hightiter antibodies IgG were detected by ELISA; FCM analyzed theratio of T cell subgroups, either group's ratio of CD4+T cell ofmice spleen cells with pVAX-S1C or pVAX-RBD immunized ishigher than that of control group; ELISPOT analyzed thesecreting condition of IFN-γafter specific antigen simulating.This result indicated the amount of spleen cell secreted IFN-γofeither experimental groups was higher than that of control group.And Statistics significance existed between pVAX-RBD and...