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A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

ABSTRACT: We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.

SUBMITTER: Bussow K

PROVIDER: S-EPMC147919 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

Büssow K K Cahill D D Nietfeld W W Bancroft D D Scherzinger E E Lehrach H H Walter G G

Nucleic acids research 19981101 21

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS. ...[more]

PMID: 9776767

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Project description:BACKGROUNDEimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines.METHODSWith the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed.RESULTSAdopting the Gateway technology, a high-quality entry library was constructed, containing 9.2?×?106 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32?×?107 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite.CONCLUSIONSOur results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

Dataset Information

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

Publications

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

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