High-Throughput Antibody Profiling Identifies Targets of Protective Immunity against P. falciparum Malaria in Thailand.

Malaria poses a significant global health challenge, resulting in approximately 600,000 deaths each year. Individuals living in regions with endemic malaria have the potential to develop partial immunity, thanks in part to the presence of anti-plasmodium antibodies. As efforts are made to optimize and implement strategies to reduce malaria transmission and ultimately eliminate the disease, it is crucial to understand how these interventions impact naturally acquired protective immunity. To shed light on this, our study focused on assessing antibody responses to a carefully curated library of P. falciparum recombinant proteins (n = 691) using samples collected from individuals residing in a low-malaria-transmission region of Thailand. We conducted the antibody assays using the AlphaScreen system, a high-throughput homogeneous proximity-based bead assay that detects protein interactions. We observed that out of the 691 variable surface and merozoite stage proteins included in the library, antibodies to 268 antigens significantly correlated with the absence of symptomatic malaria in an univariate analysis. Notably, the most prominent antigens identified were P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains. These results align with our previous research conducted in Uganda, suggesting that similar antigens like PfEMP1s might play a pivotal role in determining infection outcomes in diverse populations. To further our understanding, it remains critical to conduct functional characterization of these identified proteins, exploring their potential as correlates of protection or as targets for vaccine development.

Autoantibodies against cytoskeletons and lysosomal trafficking discriminate sarcoidosis from healthy controls, tuberculosis and lung cancers.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Hypergammaglobulinemia and the presence of autoantibodies in sarcoidosis suggest active humoral immunity to unknown antigen(s). We developed a complex cDNA library derived from tissues of sarcoidosis patients. Using a high throughput method, we constructed a microarray platform from this cDNA library containing large numbers of sarcoidosis clones. After selective biopanning, 1070 sarcoidosis-specifc clones were arrayed and immunoscreend with 152 sera from patients with sarcoidosis and other pulmonary diseases. To identify the sarcoidosis classifiers two statistical approaches were conducted: First, we identified significant biomarkers between sarcoidosis and healthy controls, and second identified markers comparing sarcoidosis to all other groups. At the threshold of an False Discovery Rate (FDR) < 0.01, we identified 14 clones in the first approach and 12 clones in the second approach discriminating sarcoidosis from other groups. We used the classifiers to build a naïve Bayes model on the training-set and validated it on an independent test-set. The first approach yielded an AUC of 0.947 using 14 significant clones with a sensitivity of 0.93 and specificity of 0.88, whereas the AUC of the second option was 0.92 with a sensitivity of 0.96 and specificity of 0.83. These results suggest robust classifier performance. Furthermore, we characterized the informative phage clones by sequencing and homology searches. Large numbers of classifier-clones were peptides involved in cellular trafficking and cytoskeletons. These results show that sarcoidosis is associated with a specific pattern of immunoreactivity that can discriminate it from other diseases.

[Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates].

OBJECTIVE: To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. METHODS: Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. RESULTS: The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. CONCLUSIONS: A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

Strongyloides-Specific IgE Phage cDNA Clones and Development of a Novel ELISA for Strongyloidiasis.

Strongyloidiasis, caused mainly by the nematode Strongyloides stercoralis, is prevalent worldwide and potentially fatal in immunosuppressed patients. We report on a new IgE biomarker to diagnose Strongyloides infection. Sera from two groups infected with Strongyloides served as positive samples: Group 1A, in which infection was confirmed by stool-microscopy and/or stool-polymerase chain reaction (PCR) and was seropositive by an IgG-enzyme linked immunosorbent assay (ELISA) and an IgG4 rapid test, and Group 1B in which infection was confirmed by stool-PCR but was seronegative. Negative samples (controls) comprised infections with other parasites (Group II) and healthy donors (Group III). Immunoscreenings of an S. stercoralis complementary DNA (cDNA) library were performed, and the cDNA clone with the highest diagnostic potential (clone A133) was selected for recombinant protein production and then evaluated using IgE Western blot and ELISA. The Western blot showed that the recombinant protein (rA133) was 100% reactive with Group IA (n = 10) and Group IB (n = 5), and 96% non-reactive with Groups II and III (n = 25). Subsequently, the IgE-ELISA was developed and showed 100% diagnostic sensitivity in Groups IA (n = 32) and IB (n = 11); and 99.3% specificity in Groups II and III (n = 144). In conclusion, this study has identified rA133 as a novel recombinant protein with potential diagnostic value, and that the IgE-ELISA incorporating this protein may be useful for patient diagnosis and epidemiological studies.

Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates / 中国血吸虫病防治杂志

Objective To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. Methods Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. Results The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. Conclusions A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

Novel T7 Phage Display Library Detects Classifiers for Active Mycobacterium Tuberculosis Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and transmitted through inhalation of aerosolized droplets. Eighty-five percent of new TB cases occur in resource-limited countries in Asia and Africa and fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays. Currently, diagnosis relies on the demonstration of the bacterium in clinical specimens by serial sputum smear microscopy and culture. These methods lack sensitivity, are time consuming, expensive, and require trained personnel. An alternative approach is to develop an efficient immunoassay to detect antibodies reactive to MTB antigens in bodily fluids, such as serum. Sarcoidosis and TB have clinical and pathological similarities and sarcoidosis tissue has yielded MTB components. Using sarcoidosis tissue, we developed a T7 phage cDNA library and constructed a microarray platform. We immunoscreened our microarray platform with sera from healthy (n = 45), smear positive TB (n = 24), and sarcoidosis (n = 107) subjects. Using a student t-test, we identified 192 clones significantly differentially expressed between the three groups at a False Discovery Rate (FDR) <0.01. Among those clones, we selected the top ten most significant clones and validated them on independent test set. The area under receiver operating characteristics (ROC) for the top 10 significant clones was 1 with a sensitivity of 1 and a specificity of 1. Sequence analyses of informative phage inserts recognized as antigens by active TB sera may identify immunogenic antigens that could be used to develop therapeutic or prophylactic vaccines, as well as identify molecular targets for therapy.

Identification of target proteins of clinical immunity to Plasmodium falciparum in a region of low malaria transmission.

The target molecules of antibodies against falciparum malaria remain largely unknown. Recently we have identified multiple proteins as targets of immunity against Plasmodium falciparum using African serum samples. To investigate whether potential targets of clinical immunity differ with transmission intensity, we assessed immune responses in residents of low malaria transmission region in Thailand. Malaria asymptomatic volunteers (Asy: n=19) and symptomatic patients (Sym: n=21) were enrolled into the study. Serum immunoreactivity to 186 wheat germ cell-free system (WGCFS)-synthesized recombinant P. falciparum asexual-blood stage proteins were determined by AlphaScreen, and subsequently compared between the study groups. Forty proteins were determined as immunoreactive with antibody responses to 35 proteins being higher in Asy group than in Sym group. Among the 35 proteins, antibodies to MSP3, MSPDBL1, RH2b, and MSP7 were significantly higher in Asy than Sym (unadjusted p<0.005) suggesting these antigens may have a protective role in clinical malaria. MSP3 reactivity remained significantly different between Asy and Sym groups even after multiple comparison adjustments (adjusted p=0.033). Interestingly, while our two preceding studies using African sera were conducted differently (e.g., cross-sectional vs. longitudinal design, observed clinical manifestation vs. functional activity), those studies similarly identified MSP3 and MSPDBL1 as potential targets of protective immunity. This study further provides a strong rationale for the application of WGCFS-based immunoprofiling to malaria vaccine candidate and biomarker discovery even in low or reduced malaria transmission settings.

A new immunoreactive recombinant protein designated as rBoSA2 from Babesia ovis: Its molecular characterization, subcellular localization and antibody recognition by infected sheep.

Ovine babesiosis, caused by the intra-erythrocytic protozoan parasite Babesia ovis, is an infectious and economically important tick-borne disease of sheep. Diagnostic testing is an essential tool used for the control of the disease. In order to identify and characterize the immunoreactive proteins which are useful in serological diagnosis of the disease, a complementary DNA (cDNA) expression library was constructed from B. ovis merozoite mRNA. A cDNA clone designated as BoSA2 was identified by immunoscreening of a cDNA library using immune sheep serum. The sequence of the BoSA2 cDNA had a partial open reading frame of 1156 nucleotides encoding a polypeptide of 384 amino acid residues. Theoretical molecular mass for the mature protein was 43.5 kDa. The sequence of the BoSA2 was inserted into the expression vector pGEX-4T-1 and then expressed in Escherichia coli DH5α cells as a glutathione S-transferase (GST)-tagged fusion protein. This recombinant fusion protein (rBoSA2) was purified by GST-affinity chromatography. Immunoreactivity of the rBoSA2 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using the sera from the animals naturally and experimentally infected with B. ovis. ELISA results demonstrated that this antigen was useful for the diagnosis of ovine babesiosis. The localization of the BoSA2 protein was shown in and on the parasite and in the cytoplasm of the infected erythrocyte by confocal laser microscope. To our knowledge, rBoSA2 is the second immunoreactive recombinant protein of B. ovis until the present.

Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens.

Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB). No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL) cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

Identification of novel tumour-associated antigens in canine mammary gland tumour.

UNLABELLED: Canine mammary gland tumour (MGT) is the most common neoplasm in female dogs and has similar biological characteristics to human MGT. Spontaneous canine MGT is a more attractive clinical model in oncological research than that of the murine experimental model. Tumour-associated antigens (TAAs), which are produced in tumour cells, are applied as tumour markers, tumour vaccine antigens and molecular targets of therapeutic drugs. In this study, we have primarily identified 13 different TAAs of canine MGT by serological immunoscreening of cDNA expression library. The results of serological mini-arrays of identified antigens showed that CCDC41 antigen specially reacted with 35% of sera from MGT-dogs and did not react with control sera. We also found that HSPH1 mRNA expression levels increased significantly in MGT tissues. These findings will contribute to the development of diagnostic technologies and translational target therapies for dogs. CLINICAL RELEVANCE: HSPH1, which is strongly expressed in the tumour tissue, will be a possible vaccine antigen of canine MGT.

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.

Expression and characterization of α-methylacyl CoA racemase from Anisakis simplex larvae.

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as α-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40℃) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Expression and Characterization of alpha-Methylacyl CoA Racemase from Anisakis simplex Larvae

Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as alpha-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40degrees C) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae.

Construction and immunoscreening of cDNA library of Armillifer agkistrodontis nymphs / 中国地方病学杂志

Objective To construct cDNA entry library and cDNA expression library of Armillifer agkistrodontis (A.) nymphs and make a preliminary immunoscreening for the cDNA expression library.Methods The nymphs were collected from the Kunming mice infected experimentally with A.agkistrodontis eggs and the total RNA were extracted from the nymphs using TRIzol Reagent.After purifying the mRNA,the synthesized cDNAs were cloned into the donor vector pDONR222 by BP reaction of Gateway technology and the recombinants were transformed into the DH10B cells by electroporation,the cDNA entry library was obtained.Next,the expression vector pDEST17 was ligated with entry clones by LR reaction,and the recombinants were transformed into the BL21 (DE3) cells.Hence,the cDNA expression library was constructed.Then,the expression library was immunoscreened with the mixed sera of mice infected with A.agkistrodontis,and the insertions of positive clones were sequenced.After that,the open reading frame(ORF) of positive slone sequence,the homology of the screened genes and their encoded proteins were analyzed by Finder and BLAST (basic local alignment search tool) program of National Center of Biotechnology Information(NCBI),and the discovered new genes were submitted into the GenBank.Besides,the physico-chemical properties,secondary structure and B cell epitopes of encoded proteins were also analyzed by bioinformatics software.Results The average titer and total clones of the cDNA entry library were 1.45 × 105 CFU/ml(colony-forming unit,CFU) and 1.74 × 106 CFU,respectively,and the range of fragment length of the inserted cDNA was between 0.2-4.0 kb,with an average of 1.4 kb.The total clones of cDNA expression library were 1.00 × 105 CFU,and the fragment length of the inserted cDNA was between 0.3-2.2 kb,with an average of 1.0 kb.Five positive clones,coded S1,S5,A1,D1 and F1,respectively,were obtained through preliminary immunoscreening.The sequence and homology of the five positive clones were sequenced and analyzed by BLAST program.No significant similarities were found in pentastomida species,which meant that they were all novel genes of A.agkistrodontis.The gene sequences were submitted to GenBank,with the accession number from JQ180451 to JQ180455.Also,results obtained by bioinformatics software showed that the predictive encoding proteins were all potential to be valuable recombinant diagnostic antigens.Conclusions The cDNA library of A.agkistrodontis nymphs is successfully constructed,and five new genes of A.agkistrodontis are discovered.The establishment of cDNA library and the discovery of the new genes will lay a foundation for further studying the gene functions and screening the immunodiagnostic antigens.

Identification of antigenic proteins in Trichomonas vaginalis.

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

Identification of Antigenic Proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage lambda Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, alpha-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

Characterization of a clone from an adult worm cDNA library selected with anti-Schistosoma mansoni human antibodies dissociated from immune complexes: a preliminary report

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.

Study on construction of cDNA library of the treated changliver cell and quality analysis.

The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 10(10) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. These results showed that the Changliver cell cDNA library had an excellent quality and lay foundation for screening whole long cDNA of related genes.

Construction of cDNA library of the treated Changliver cell and quality analysis / 新乡医学院学报

Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

Immunoscreening and Sequence Analysis of a cDNA Library of Adult Trichinella spiralis / 中国寄生虫学与寄生虫病杂志

Objective To obtain an antigenic gene of adult Trichinella spiralis. Methods cDNA library of the adult Trichinella spiralis was screened using the sera of immunized and infected rabbits. The gene sequence was analyzed by DNAstar software and GenBank database. Results Nine positive clones were identified by immunoscreening. The clone Ts87 was sequenced and a cDNA with 1 172 bp full length was obtained using 5′ RACE technique, encoding 347 amino acids. Some possible antigen epitopes were predicted. Conclusion A novel antigenic gene of Trichinella spiralis was obtained.