Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-04-19
2004-07-20
Ibrahim, Medina A. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S288000, C800S298000, C435S320100, C435S069100, C435S419000, C435S468000, C536S023700
Reexamination Certificate
active
06765127
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to the field of proteins associated with the peritrophic membranes of insects. More particularly, the invention pertains to a novel invertebrate intestinal mucin cDNA and related products and methods.
BACKGROUND OF THE INVENTION
Vertebrate epithelial organs are covered, throughout the body, with a mucus lining, which serves as a selective physical barrier between extracellular contents and the epithelial cell surface. The mucus lining, especially in the gastrointestinal tract, is highly resistant to various digestive enzymes and provides protection and lubrication for the underlying cells. The protective functions of the mucosal layer are largely dependent upon heavily glycosylated proteins known as mucins. Mucins play an active role in preventing bacterial, viral, and other pathogens from interacting with vertebrate intestinal epithelia.
Mucins are highly O-glycosylated proteins. Carbohydrate moieties on mucins commonly account for more than 50% of the protein by weight. The biochemistry and molecular biology of mucins from vertebrates ha been broadly investigated, with human epithelial mucins being the most extensively studied. Several mucins from humans and other vertebrates have been completely or partially sequenced, and this has contributed to a greater understanding of their structure and function. Full cDNA sequences for human mucin MUC1, MUC2, and MUC7, have been obtained. In addition, mucins from other vertebrates, including mouse MUC-1, rat ascites sialo-glycoprotein-1, canine tracheobronchial mucin, bovine submaxillary mucin-like protein, and frog IIM-A.1, have also been fully sequenced by cDNA cloning.
Studies on invertebrate mucins are very limited in comparison with vertebrate mucins.
Drosophila melanogaster
“glue proteins” from salivary glands have structural characteristics of mucin-like proteins. These “glue protein” have been sequenced but their function has not been fully determined. Mucin-like proteins have also been reported in protozoans. A secretory mucin involved in maintaining the cohesiveness of a clutch of a squid egg-mass formation was identified from that animal's nidamental gland. A glycoprotein from
Drosophila melanogaster
cultured cells was reported to be a mucin-like protein. Recently, a membrane-associated mucin from the hemocytes of
Drosophila. melanogaster
was identified, and a cDNA for the mucin was subsequently cloned. However, to date, there have been no reports on mucins identified from invertebrate digestive tracts.
Part of the reason for this may be that insects do not possess a mucus layer lining the digestive tract and/or other epithelial cells, as do vertebrates. The digestive tract in insects is commonly lined with an invertebrate-unique structure, the peritrophic membrane (PM). PMs are non-cellular matrices composed primarily of chitin, protein, and glycoproteins. PMs demonstrate a protective function similar to the mucus layer in vertebrates (e.g. a selective barrier protecting the digestive tract from physical damages and microbial infections).
Although there are few studies on the interaction between microbial pathogens and PMs, these structures are proposed to serve as a physical barrier to invasion or infection by pathogenic microorganisms. The chitin component of PMs is normally present as a network of chitin fibrils in which proteins and glycoproteins are present. The chitin can be a potential target substrate for intestinal pathogens. This was demonstrated through the degradation of chitin in the PM by a pathogen-encoded chitinase allowing an avian malaria parasite to overcome its mosquito vector intestinal PM barrier and infect the vector itself.
Proteins are the major PM component; however, their functions in the PM are unknown. Studies on the PM proteins are limited to analyses of the amino acid composition of total PM proteins and PM protein profiles as determined by electrophoresis. The only PM protein characterized to date, peritrophin-44, was isolated from
Lucille cuprina
larvae, but its biological function is not fully understood. To date, studies on the interaction of PM proteins with microbial pathogens are limited to the effect of a baculovirus enhancin on lepidopteran PM proteins.
Previous studies have demonstrated that a
Trichoplusia ni
granulosis virus (TnGV) encodes an enhancin protein, a viral enhancing protein, that was identified as a metalloprotease. Enhancin degrades high molecular weight PM proteins in vivo and in vitro. In addition, the protein degradation initiated by these enhancins is correlated with the disruption of the structural integrity of the PM thereby “enhancing” viral infection. It was recently demonstrated that enhancin could degrade high molecular weight PM proteins from several lepidopterous species; however, the chemical nature and function of these proteins in baculovirus pathogenesis were previously unknown.
With a more complete knowledge of the proteinaceous components of the PM, and particularly the mucin-like proteins it will be possible to use that information to enhance the effectiveness of bio-engineered pesticides, recombinant viral vectors, enhance the defenses of transgenic plants, or protect insect vectors susceptible to attack by organisms utilizing enhancin or enhancin-like enzymes.
SUMMARY OF THE INVENTION
Briefly stated the current invention represents the disclosure of a novel intestinal insect mucin comprising two nearly identical isoforms, IIM14 and IIM22 respectively. The proteins are identical except for slightly different peptide length in some repetitive regions, which is common in mucin proteins. This IIM protein has been identified and cloned from
T. ni
larva. Its cDNA and amino acid sequences have been determined and are disclosed. The IIM protein has an approximate molecular mass of 400 kDa. These sequences are useful for the production of transgenic or recombinant vectors including viral, microorganism, cell, plant, or animals, wherein the virus, microorganism, cell, plant, or animal is the product of an insertion of a gene expression vector including a DNA that encodes an IIM protein sequence. Thereafter the engineered host of the IIM DNA sequence is capable of expressing said IIM protein in a functional form. One easily used host is the bacteria is
Escherichia coli.
Also useful is a purified and isolated recombinant DNA sequence comprising a DNA sequence that codes for an IIM protein. The recombinant DNA sequence used can be a cDNA sequence for either IIM14 or IIM22, SEQ. ID.'s No. 1; and 2 respectively. The current invention also provides for the use of the purified or recombinant proteins, IIM14 or IIM22, SEQ. ID.'s 3 or 4 respectively.
With the cloned IIM sequence it is possible to prepare an IIM protein or peptide by transforming a host cell with an expresssion vector comprising a promoter operatively linked to a nucleotide sequence which codes for a fusion protein wherein said fusion protein comprises a first protein or peptide fused directly or indirectly with a transfer molecule (glutathione-S-transferase), wherein said first protein or peptide is a predetermined protein or peptide of a
T. ni
IIM protein. Then culturing the host cell under conditions such that the fusion protein is expressed in recoverable quantity. When harvesting the protein or peptide the cells must be collected, isolated, lysed, and the fusion protein purified from the cytosol.
A gene expression vector containing a recombinant DNA sequence encoding a
T. ni
IIM protein sequence can also be constructed with this technology. This is accomplished through the use of a recombinant plasmid adapted for insertion into and transformation of bacteria or transgenic plants such that these hosts can express either the IIM protein or antibodies to disrupt pertrophic membrane function and formation in larval pests. The antibodies expressed by the plant could bind to the mucin or its ligand or portions the IIM protein could be expressed by the plant to result in competive binding with the larvae's expressed mu
Granados Robert R.
Wang Ping
Boyce Thompson Institute for Plant Research Inc.
Brown & Michaels PC
Ibrahim Medina A.
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