The Texas A&M University System

American Cyanamid Company

Max D Summers

Gale E Smith

Bruce Christian Black

James Y Bradfield

Sharon C Braunagel

Larry L Keeley

Tao Hong

Christian E Oker-Blom

Provided are hydrophobic targeting sequences, which may serve to target heterologous proteins to a variety of cellular membranes. In particular, the structural components of the nuclear envelope, or those components which become nucleus-associated, may be targeted with the sequences provided. Also provided are methods of targeting heterologous proteins to particular membranes, and the use of these targeted proteins in therapeutic, diagnostic and insecticidal applications.

Unique nucleotide and amino acid sequence and uses thereof

Seven heterologous signal sequence are described for use with genes for insect controlling proteins, such that when the signal sequence and protein genes are inserted into an insect virus, that virus demonstrates an earlier onset of morbidity than a wild-type insect virus which lacks the gene for the insect controlling protein.

Heterologous signal sequences for secretion of insect controlling

The engineering of foreign vertebrate gene constructs by recombinant DNA techniques for the more efficient processing and secretion of foreign genes in insect systems is done by replacing the foreign vertebrate protein signal peptide sequences with protein signal sequences from insect cell secreted proteins. A baculovirus expression vector system is constructed wherein the natural signal DNA peptide sequence associated with the desired foreign gene, such as CD4 (T cell surface protein T4) is replaced by the signal DNA sequence from the insect signal peptides coding for the cuticle gene or adipokinetic hormone.

Method to improve the efficiency of processing and secretion of foreign

This invention relates to specifically designing and genetically engineering recombinant baculovirus for producing, in a compatible insect system, a desired protein, virus, protein hybrid, or virus hybrid. In particular aspects, this invention relates to the use of different baculovirus promoters for the ultimate purpose of constructing a recombinant baculovirus designed for the investigator's specific need. For example, the recombinant baculovirus of this invention can be designed to produce a viral pesticide. This invention also describes the construction of a genetically engineered virus or virus hybrid (e.g. animal or human pathogen) which is not capable of replicating itself but is essentially identical to the authentic pathogen in terms of structure and antigenicity. This baculovirus is constructed such that the non-structural viral genes are truncated, mutated or both and are located 3' and directly under the control of an early baculovirus gene promoter and the structural viral genes are located 3' and directly under the control of a late baculovirus gene promoter. This genetically engineered baculovirus is therefore capable of temporal regulation and successive synthesis of non-structural and structural proteins. The truncated or mutated non-structural viral genes creates the non-replicative aspect of this invention. Since the genetically produced virus or virus hybrid is essentially identical to the authentic pathogen, the product is thereby highly antigenic and potent in terms of efficacy and efficiency. This invention enables the design and constructure of a virus particle or virus hybrid with specific antigenic properties which further allows for the safe and inexpensive production of vaccines or diagnostics.

Baculovirus dual promoter expression vector

Insect signal sequences useful to improve the efficiency of processing

A baculovirus expression vector system is constructed wherein the natural signal DNA peptide sequence associated with the desired foreign gene, such as CD4 (T cell surface protein T4) is replaced by the signal DNA sequence from the Lepidopteran adipokinetic hormone (AKH) precursor signal peptides. The exemplary Manduca sexta AKH signal sequence is represented as follows: ##STR1## .

Lepidopteran AKH signal sequence

A method for producng a recombinant baculovirus expression vector, capable of expressing a selected gene in a host insect cell, is disclosed. The method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. A recombinant transfer vector is prepared by inserting said DNA fragment into a cloning vehicle and thereafter inserting a selected gene into the thus modified cloning vehicle such that it is under the transcriptional control of the polyhedrin promoter. The recombinant transfer vector is contacted with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome. The resultant recombinant baculovirus is then used to infect susceptible insects or cultured insect cells and the protein product from the incorporated selected gene is produced from the infection.

Method for producing a recombinant baculovirus expression vector

A method for producing a recombinant baculovirus expression vector, capable of expression of a selected gene in a host insect cell, is disclosed. The preferred method involves cleaving baculovirus DNA to produce a DNA fragment comprising a polyhedrin gene or portion thereof, including a polyhedrin promoter. A recombinant shuttle vector is prepared by inserting said DNA fragment into a cloning vehicle and thereafter inserting a selected gene into the thus modified cloning vehicle such that it is under the transcriptional control of the polyhedrin promoter. The recombinant shuttle vector is contacted with a baculovirus DNA so as to effect recombination and incorporation of the selected gene into the baculovirus genome.

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Bryan, TX, United States of America

Max D Summers