Cloning, Expression And Preliminarily Application Of Several Antigenic Determinant Genes Of Human Cytomegalovirus

Human cytomegalovirus (HCMV) ,a beta herpesvirus,which has duplex DNA structure ,is ubiquitously distributed in human populations.Although rarely pathogenic in immunocompetent individuals,the virus poses a significant cause of morbidity and mortality in organ allograft ,AIDS patients, immunosuppression patients and so on.Pregnant women are also a risk group for this virus as HCMV is the most common cause of congenital infection.Sience the infections with HCMV either are asymptomatic or are accompanied by symptoms not specific for HCMV(such as fever and leucopenia),laboratory techniques are the sole means of diagnosing HCMV infection. So it has significant sense to build up a fast, sensitive and specific laboratory method for the diagnosis of HCMV, then doctors can take therapeutic measures in the early period of the infection which have positive meaning in aristogenesis and preventing the virus's conveying.Diagnosis of HCMV infection can be obtained by direct demonstration of the virus or virus components in pathological materials or indirectly through serology. The most direct methods include culture of the virus or detection of viral components ,like viral DNA,RNA and antigens in body fluids or tissue biopsy specimens.Diagnosis of primary HCMV infection is exclusively accomplished by serological methods.Serologic assays are widely used for donor selection and to support the diagnosis of HCMV in the host and to determine whether it is an active or latent infection.Although it indirectly reflects viral activity ,serology provides a cheap alternative method that can readily be automated for routine use.HCMV-specific immunoglobulin M(IgM) is a sensitive and specific indicator of primary HCMV infection in immunocompetent subjects,while it is very often produced during viral reactivation in transplant recipients, ,AIDS patients and so on.In current serologic assays complex viral lysates are commonly used.However, detection of HCMV-specific IgMvaries widely,and poor agreement has been found among the results obtained with different commercially available kits.The use of these viral lysates has disadvantages because they consist of many viral antigens whose exact compositions are difficult to standardize.Preparation of lysates requires culture of HCMV in fibroblasts,resulting in potential contamination with cellular proteins. Since many transplant recipients may temporarily develop autoantibody responses ,a false-positive reactivity may result.Another problem can arise,since herpesviruses share multiple protein homologues,which can give rise to cross-reactivity in assays based on complex viral lysates.In order to overcome these problems the viral lysate should be replaced by a defined antigen preparation,preferably consisting of a combination of HCMV-specific and immunodominant antigens,in order to achieve the highest sensitivity and specificity.Recombinant proteins from genetic engineering can be used to detect antibodies to the parent protein.Combinations of such defined immunodominant proteins or peptides may ideally be suited as replacements for complex protein-antigen mixtures. Some antigenic determinants were investigated to seek immunogenic and protective for potential diagnosis.gp52(UL44),pp150 (ppUL32),pp65 (ppUL83),ppUL80a are all immunogenic antigen determinants with most conservative DNA sequences,and can recognize the HCMV-IgM in the serum specifically.In this study,these five antigenic determinant genes(ppl50 aa595-6149 (A) ,ppl50 aa 1006～1042 (B),gp52 aa202～434 (C), pp65 aa297～510 (D) ,ppUL80a aa117～373 (E)) have been amplified successfully from HCMV genome and then the first three fractions were recombinated into a new fraction(F) by Overlap PCR.Then the three fractions(D,E,F)were cloned into a cloning vector,and the DNA sequencing proved that the fractions amplified by PC R are totally correct.The correct gene fragments were cloned into a prokaryotic expression vector to generate fusion proteins named GST- ppUL83, GST- ppUL80a, GST-LINK respectively, which were induced by IPTG and well expressed in E coli.The HCMV immuno-specificity of these three fusion protein weredetected by western-blotting.All three recombination proteins could be specially recognized by HCMV IgM sera,however the vector protein GST could not react with the infected sera,indicating the ideal antigenicity of the three recombination protein and the application of the HCMV diagnosis.According to the different bio-characteristic,different purification methods were used to acquire purified antigen,and the three antigens had been purified successfully. Time- resolved fluoroimmunoassay(TRF1A) is a kind of new non- radio marked technique rised from 80's last century. It makes use of the special fluorescence characteristic of the lanthanum series and their chelate to mark antigens,antibodies,probes of nucleic acid and so on.After the reaction system(for example:antigen-antibody reaction,hybridization of nucleic acid probes) occures ,determinant the fluorescence intensity of the reaction product by the TRFIA tester. According to the specific value of the outcome fluorescence intensity and the opposing fluorescence intensity, we can decide the concentration of the analyte in the reaction system. The TRF1A dexterously utilize the special fluorescence characteristics of the lanthanum series :first,the decay time of the fluorescence of the lanthanum series' chelate is very long, which is 10~3-10~6 times of traditional fluorescences ;second:The Stokes bias between the excitation light and the emission light can amount to 290 nm, while the common fluorescences' Stokes bias is only 28 nm. Thus it overcomes the weakness of the common fluorescence examination :strong intensity of the background fluorescence and interference, thusdistinguish the strong particularity fluorescence and the background fluorescences .It can almostly remove the interference of the background fluorescence, raising the sensitivity of the examination. Compared with the common emanation such as radioimmunoassay(RIA),enzyme-immunoassay(EIA),chemiluminescence immune assay(CLIA) ,the TRFIA has some advantages ,for example,the high sensitivity,the simple operation process,the stabilization of the tracer, the wide extent of the standard curve,no radioaction contaminate,the little interference in the bioactivity of the marked mass,and so on.Now,TRFIA is one of the excellent analysis methods in biological investigation and clinical ultramicro-examination of biochemistry.To check whether the three pured purpose proteins contain the antigenic determinant that can react specially with the HCMV lgM in the sera,we make use of the purpose proteins and the complete virus antigen of schizolysis(CMV grade2 antigen of Microbix Biosystems company), utilizing the TRFIA method to detect the serums which we have already known the positive or negative of HCMV IgM, then the results were analyzed by Linear Correlation. The analysis result demonstrat that the result of the three purpose proteins used prospectively has positive correlation with the result of CMV grade2 antigen, but the result is not very well.While the result of the three purpose proteins used together has fine positive correlation with the result of CMV grade2 antigen, r_s=0.989. The main reason may be although the proteins we chosed in this experiment:gp52, pp150, pp65, ppUL80a are all proteins with strong immunogenicity, conservative sequence of DNA and have the ability to react with HCMV IgM sensitivity and specificity, they can only react with HCMV IgM of particular stage of HCMV infection. The analysis of the humoral immune response elicited during natural infection has repeatedly shown that the basic phosphoprotein of 150 KDa encoded by UL32(ppUL32) and localized in the viral tegument is highly immunlgenic and is recognized by sera from nearly 100% of the HCMV-seropositive subjects tested.In this molecule at least two epitopes have been identified and shown to react efficiently with human immunolobulins. In particular, the analysis of several ppUL32 fusion proteins showed that a region localized in the last 43 amino acids at the carboxy terminus of the molecule(amino acids 1006 to 1048)reacts with more than 80% IgM-positive serum samples. When chemically synthesized, antother region localized between anino acids 595 and 614 gave a positive reaction with almost 100%IgG-positive serum samples. When it is expressed as recombinant protein,this region also reacted efficiently with HCMV=specific IgM.The two coding regions were recently fused together and were shown to produce a double-epitope fusion protein which can replace the entire p150 molecule in its lgM-binding ability. Another HCMV protein which reacts very well with IgM is ppUL44, the nonstructural DNA-binding protein of 52 kDa. The carboxy-terminal part of th molecule was chosen because it does not contain relevant amino acid sequences cross-reacting with the homologous protein of other members of the Herpesviridae family. The carboxy-terminal half of this protein was expressed and tested with human sera alone and in combination with the double-ppUL32 epitope fusion protein. Besides ppUL32 and ppUL44, two other HCMV proteins were chosen:the major matrix protein ppUL83 and the assembly protein(ppUL80a). The first is known to induce a strong IgM response, and antibodies reacting exclusively to this protein were described during primary infection. The second protein was included in the study because in HCMV-seropositive transplant recipients undergoing viral reactivation, IgM to this protein is very often the first marker to be detected. Use the three proteins together to detect the serums which we have already known the positive or negative of HCMV IgM, the result has fine positive correlation with the result of CMV grade2 antigen,which demonstrate that the chosen proteins contains the antigenic determinants that can react sensitively and specifically with the HCMV IgM. Use the three proteins together according to the optimum to check 100 random sera, this result compared with the results of commercial kits from internal and abroad having excellent efficiency, didn't show significant difference.