Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.     

Genetic transformation of sweet orange with the coat protein gene of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> and evaluation of resistance against the virus

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.     

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).     

<Emphasis Type="Italic">Citrus psorosis virus</Emphasis> coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes

Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.     

Production of polyclonal antibodies to a recombinant coat protein of potato virus Y

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.     

Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virus

The coat protein (CP) coding regions of two Czech Potato mop‐top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV‐CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV‐specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme‐linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA‐ ELISA (indirect plate‐trapped antigen (PTA)‐ELISA).     

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D ‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM . About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Secretory expression of a novel human spermatozoa antigen in <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> and its application to a protein chip

Objective

To produce a recombinant spermatozoa antigen peptide using the E. coli: PhoA system on a protein chip for screening anti-sperm antibodies (ASA).

Results

The purity of the recombinant spermatozoa antigen exceeded 95% after two-step purification, as assessed using SDS-PAGE and HPLC. The diagnostic performance of a protein chip coated with the recombinant antigen peptide was evaluated by examining ASA in 51 infertile patients in comparison with a commercial ELISA kit. The area under the receiver operating characteristic curve (AUC) was 0.944, which indicated that the protein chip coated with recombinant spermatozoa antigen peptide was consistent with ELISA for ASA detection.

Conclusion

A recombinant spermatozoa antigen was expressed in the E. coli PhoA secretory expression system and its potential application for clinical ASA detection was validated.

     

Production of Polyclonal Antibodies Using Recombinant Coat Protein of Papaya ringspot virus and their Use in Immunodiagnosis

Production of polyclonal antibodies requires large amount of purified virus that can be avoided by the use of recombinant coat protein (CP). Recombinant CP of Papaya ringspot virus (PRSV) was thus used for the production of polyclonal antibodies as the virus purification from papaya tissues provides low virus yields. CP was expressed as a fusion protein (～72 kD) containing a fragment of E. coli maltose binding protein. Polyclonal antibodies from rabbits immunized with the fusion protein, successfully detected natural infection of PRSV in papaya and cucurbits samples collected from different locations at 1:4000 dilution in direct antigen-coated enzyme-linked immunosorbent assay.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Autodisplay of 60-kDa Ro/SS-A antigen and development of a surface display enzyme-linked immunosorbent assay for systemic lupus erythematosus patient sera screening

Enzyme-linked immunosorbent assay (ELISA) is a common tool to test human sera on an antibody reaction against a specific antigen. The 60-kDa Ro/SS-A antigen for autoantibodies can be found in sera from systemic lupus erythematosus (SLE) patients. As in the case of 60-kDa Ro/SS-A, antigens used in ELISAs are recombinantly expressed in Escherichia coli and time-consuming purification steps are needed to get the proteins. To avoid these disadvantages, 60-kDa Ro/SS-A was expressed on the surface of E. coli using autodisplay, an efficient surface display system. Cells displaying 60-kDa Ro/SS-A on the surface were applied as an antigen source instead of the purified antigen. In total, 39 patients and 30 control sera were screened on a 60-kDa Ro/SS-A antibody reaction. To eliminate antibodies against native E. coli, human sera were preabsorbed with E. coli cells prior to the assay. The new ELISA protocol (surface display ELISA [SD-ELISA]) using E. coli with autodisplayed 60-kDa Ro/SS-A showed a sensitivity of 86.67% and a specificity of 83.33% by a cutoff value of 0.28. Our results show that autodisplay provides simple, rapid, and cheap access to human antigens for an ELISA to screen human sera against specific antibody reactions.     

Serological Detection of Grapevine leafroll virus 2 Using an Antiserum Developed against the Recombinant Coat Protein†

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL‐c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP‐CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti‐MBP‐CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti‐MBP‐CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate‐trapped antigen enzyme‐linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Production of Polyclonal Antibodies to the Recombinant Coat Protein of Citrus tristeza virus and their Effectiveness for Virus Detection

Citrus tristeza virus (CTV) is distributed worldwide and causes the most economically important virus diseases of citrus. Enzyme‐linked immunosorbent assay (ELISA) and/or immunoprinting have become an indispensable tools for large‐scale diagnosis of CTV worldwide. Several CTV detection kits are commercially available, based on either polyclonal or monoclonal antibodies developed against purified virus preparations. We have developed polyclonal antibodies to recombinant p25 CTV coat proteins (rCP) and determined their effectiveness for both trapping and as the intermediate antibody in double‐antibody sandwich indirect (DASI) ELISA. The p25 coat protein gene of three CTV isolates was amplified by RT‐PCR and further cloned and expressed in Escherichia coli cells. The rCP was injected into rabbits and goats for antibody production. Western blotting assays with the rCP CTV‐specific antibodies reacted positively with the homologous and heterologous rCP of the three CTV isolates and with the corresponding native coat protein present in crude sap extracts of CTV‐infected citrus tissue, but not with extracts from healthy tissue. The rCP antibodies from goat and rabbit reacted as both plate trapping and intermediate antibodies in DASI‐ELISA, discriminating healthy and CTV‐infected citrus, with optical density (OD₄₀₅) values in the range of 0.151–2.415 for CTV‐infected samples and less than 0.100 for healthy tissue. Commercially available anti‐CTV antibodies were used as a reference. Previous reports indicate that antibodies developed to recombinant antigens, including those of CTV, may not be functional for trapping the target antigens under non‐denaturing conditions. Our results showed the feasibility of CTV antibodies developed to the rCP for use as both trapping and intermediate antibodies in DASI‐ELISA, when the recombinant antigen was fractioned with polyacrylamide electrophoresis gel and further extensively dialysed against phosphate buffer saline prior to its use as immunogen.     

Development and evaluation of a recombinant CP23 antigen-based ELISA for serodiagnosis of Cryptosporidium parvum

The CP23 gene of Cryptosporidium parvum strain isolated from Changchun in China was expressed in Escherichia coli BL21 strain and was purified as a recombinant protein. An indirect ELISA assay (CP23-ELISA) for antibody detection was established using the purified recombinant CP23 protein. Antigen coating conditions and serum dilution for the CP23-ELISA were optimized. The S/P ratio of the absorbency value was calculated in the CP23-ELISA to evaluate the serum antibody level of the field cow samples. It indicated that the CP23-ELISA assay, which was more convenient and easier to prepare than traditional methods, was a good candidate for evaluation of C. parvum exposure to domestic animal in field.     

Asia I口蹄疫vp2蛋白单克隆抗体的制备及单抗竞争ELISA方法的建立

制备Asia I口蹄疫病毒vp2单克隆抗体(mAb)并建立了单抗竞争ELISA方法。用纯化的Asia I型口蹄疫病毒vp2重组蛋白免疫BALB/c小鼠, 将免疫小鼠的脾细胞与骨髓瘤SP2/0细胞融合, 采用间接ELISA和有限稀释法筛选杂交瘤细胞。分别用ELISA、Western blotting检测mAb腹水的效价及其特异性。筛选到杂交瘤细胞2株, 腹水效价均在100×29以上; 以纯化后的Asia I型口蹄疫病毒vp2重组蛋白作为抗原, 利用Asia I型口蹄疫病毒vp2单抗酶标物建立了竞争ELISA方法用来检测Asia I型口蹄疫抗体。临床应用表明, 该方法与UBI公司的口蹄疫全病毒抗体检测试剂盒总符合率达89.0%, 和荷兰赛迪公司的口蹄疫病毒LPB-ELISA抗体检测试剂盒总符合率达86.5%。