Boyce Thompson Institute for Plant Research

Robert R Granados

Ping Wang

Yoshifumi Hashimoto

Gary W Blissard

Guangyun Lin

Guoxun Li

Kevin A McKenna

Cell lines that are commonly used for protein expression are engineered to include genes that encode suppressors of apoptosis (SA). Insect cell lines expressing these SA genes are resistant to apoptosis or programmed cell death, and express certain recombinant proteins at increased levels. These cell lines also have increased resistance to many types of stress. Because some of the SA proteins inhibit apoptosis in a wide spectrum of organisms, these genes may be inserted into other plant or animal cell lines for a variety of purposes involving resistance to apoptosis or resistance to stress.

Stable cell lines resistant to apoptosis and nutrient stress and methods of making same

Improved insect cell lines, two of which are designated H5CL-B and H5CL-F (both of which are derived from the parental cell line BTI-TN-5B1-4, ATCC CRL 10859), possess the properties of increased production of baculovirus particles, increased expression of foreign proteins using a baculovirus expression system, and increased resistance to cell culture stress, relative to the parental cell line.

Clonal cell lines derived from BTI-TN-5B1-4

Recent advances of the research on synergistic effect of a mixed baculovirus infection demonstrated the presence of viral molecules enhancing the early event of infection. The enhancins from thewas identified to have such a function, i.e., disrupting the structural integrity of peritrophic membrane of midgut oflarva. The enhancin gene was ligated downstream of the CaMV 355 promoter of a binary vector pBI121. With a drug resistant gene, the gene was introduced to a piece of tobacco leaf, fromcv. Havana SR1. We screened 11 regenerated plants out of 37 by feeding tobacco powder mixed in artificial diet, to 3rd instarlarvae. The larval stage was usually delayed from 1 to 3 days in comparison of that of control larvae. The larvae did not pupate normally. The larvae showed irregular morphology of half larva and half pupae, suggesting a hormonal disturbance caused by the transgenic tobacco. The introduction of an enhancin gene into plants is an effective method of protecting them from insects due to the disruption of their normal life cycle.

Method of protecting plants by introducing a gene coded for a protein which enhances virus infection of host insects

The invention represents the disclosure of an insect intestinal mucin (IIM) protein. The IIM protein was been identified and cloned using larva. The cDNA and amino acid sequences have been determined and are disclosed. These sequences are useful for the production of transgenic cells, including plant cells, having insecticidal activity.

Invertebrate intestinal mucin cDNA and related products and methods

This disclosure presents the establishment of new cell lines from eggs, in a commercial serum-free medium. These new cell lines were screened with wild-type multiple nuclear polyhedrosis virus (AcMNPV) and single nuclear polyhedrosis virus (TnSNPV). In addition, selected cell lines from the initial screenings with the native baculoviruses, were further tested for their capacity to express recombinant proteins.

Establishment of Trichoplusia ni cell lines in serum-free medium for recombinant protein and baculovirus production

The invention represents the disclosure of a novel insect intestinal mucin comprising two nearly identical isoforms, IIM14 and IIM22 respectively. These isoforms of the IIM protein have been identified and cloned using,larva. The cDNA and amino acid sequences have been determined and are disclosed. Both IIM isoforms have an approximate molecular mass of 400 kDa. These sequences once disclosed 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. 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 IIM isoform IIM14 or IIM22, SEQ. ID.'s No. 1; and 3 respectively. The current invention also provides for the use of the purified amino acid sequences of IIM isoforms IIM14 or IIM22, SEQ. ID.'s 2 or 4 respectively. With this knowledge of the proteinaceous components of the PM, and particularly the mucin-like proteins it will be possible 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.

This disclosure relates to an isolated and cloned DNA from a granulovirus virus which comprises an amino acid sequence of the vital gene encoding a polypeptide isolated from occlusion bodies of certain baculoviruses and which polypeptide possesses the biological activity of enhancing baculovirus infectivity. Such proteins termed herein as 'enhancins' are found within the viral occlusion body, have a disruptive effect on the insect peritrophic membrane (PM) proteins, and/or interact with the midgut epithelium in such a manner as to permit the increased adsorption, penetration and uptake of virus particles by midgut cells with a concomitant increase in host mortality. Disclosed herein is a recombinant DNA sequence which codes for the enhancin protein of the Helicoverpa armigera granulovirus virus. The DNA sequence is shown in SEQ. ID. NO.: 1 and the open reading frame is shown in SEQ. ID. NO.: 1: base pairs 271-2976. The amino acid sequence of the enhancin protein is shown in SEQ. ID. NO.: 2.

DNA sequence coding for enhancin polypeptide which enhances virus

This disclosure presents the establishment of a new cell line from Pseudaletia unipuncta embryos. This cell line demonstrated the ability to produce high numbers of baculoviruses in cell culture. These virus particles are found internally in the cells in occlusion bodies. In the study of Pseudaletia unipuncta two baculoviruses were found to infect this species: P. unipuncta nuclear polyhedrosis virus (PuNPV), and P. unipuncta granulosis virus (PuGV). In addition, the cell line was also selected and cultured for its ability to grow in suspension while maintaining high levels of OB production.

Establishment of new cell lines from Pseudaletia unipuncta with

Cloning and sequencing certain baculovirus genes encoding polypeptides termed enhancins. The polypeptides are isolated from the occlusion body of certain baculoviruses such as Trichoplusia ni granulosis virus and Pseudaletia unipuncta granulosis virus, Hawaiian strain. The polypeptides have the ability of enhancing the infectivity of baculoviruses and are useful ingredients of pest control compositions.

Gene coded for a polypeptide which enhances virus infection of host

Normally anchorage-dependent insect cell lines are adapted to replicate under suspension conditions by addition of heparin to the culture medium and selection for resulting suspension-tolerant cells.

Method of adapting anchorage-dependent cell lines to suspension

An insect cell line has been established and characterized, derived from embryonic tissue (BTI-TN-5B1-4, ATCC CRL 10859) of Trichoplusia ni (cabbage looper). The line is susceptible to various baculoviruses, including TnSNPV and AcMNPV, and may be used to replicate such viruses for use as insecticides or otherwise.

Trichoplusia ni cell line which supports replication of baculoviruses

Two new insect cell lines have been established and characterized; the cell lines were derived from midgut (BTI-TN-MG1, ATC CRL 10860), and embryonic tissue (BTI-TN-5B1-4, ATC CRL 10859) of Trichoplusia ni (cabbage looper). The lines are susceptible to various baculoviruses, including TnSNPV and AcMNPV, and may be used to replicate such viruses for use as insecticides or otherwise.

Cell line isolated from larval midgut tissue of Trichoplusia ni

Nuclear polyhedrosis viruses, for example, Autographa californica nuclear polyhedrosis virus (AcMNPV), useful in the control of lepidopterous larvae such as the larvae of the cabbage looper Trichoplusia ni, have been found to have enhanced infectivity when mixed with certain proteins obtained from the granulin fraction of Trichoplusia ni granulosis virus (TnGV) or Heliothis armigera granulosis virus (HaGV), and from the polyhedrin fraction of AcMNPV viruses. The proteins from the TnGV granulin fraction have molecular weights of about 101 and about 104 Kd. The enhanced infectivity is correlated to biochemical and structural changes in the T.ni peritrophic membrane.

Baculovirus proteins and viral pesticides containing same

Nuclear polyhedrosis viruses, for example, Autographa californica nuclear polyhedrosis virus (AcMNPV), useful in the control of lepidopterous larvae such as the larvae of the cabbage looper Trichoplusia ni, have been found to have enhanced infectivity when mixed with certain proteins obtained from the granulin fraction of Trichoplusia ni granulosis virus (TnGV) or Heliothis armigera granulosis virus (HaGV), and from the polyhedrin fraction of AcMNPV viruses. The proteins from the TnGV granulin fraction have molecular weights of about 101 and about 104 kd. The enhanced infectivity is correlated to biochemical and structural changes in the T.ni peritrophic membrane.

Growing community of inventors

Ithaca, NY, United States of America

Robert R Granados