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Methods of diagnosing cervical cancer

A kit and papilloma virus technology, applied in the field of HPV E6 protein composition, can solve the problems of expensive, difficult to detect E6 protein, and inability to recognize E6 protein

Inactive Publication Date: 2011-04-13
生命树股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While the assays described are helpful in differentiating oncogenic from non-oncogenic infections, these assays are quite expensive to perform and require trained technicians to perform PCR and / or photometric detection
In addition, PCR has a natural rate of false positives that may require further testing and unnecessary manipulation
Since HPV's carcinogenicity has been shown to be protein-based, early detection of HPV DNA or RNA may lead to unnecessary medical steps that the body's immune system can naturally resolve
[0008] There are many difficulties in the detection of oncogenic HPV in human samples (such as tumor samples) by traditional methods
For example, detection of E6 protein with antibodies is difficult because E6 produced in human cells contains various structural changes (such as disulfide bonds and phosphate groups), making wild-type E6 protein produced in bacterial systems or chemically synthesized E6 Peptides do not recognize E6 protein in human cells
Moreover, since oncogenic E6 proteins have no shared epitopes that distinguish them from non-oncogenic E6 proteins, a single antibody cannot be used to detect all oncogenic E6 HPV strains

Method used

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Experimental program
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Embodiment 5

[0097] Example 5 lists representative PDZ domain sequences cloned in a vector (PGEX-3X vector) for the generation of GST-PDZ fusion protein (Pharmacia). An extended list of PDZ domains cloned in pGEX vectors for the generation of GST-PDZ fusion proteins is given in US Patent No. 09 / 724553.

[0098] As discussed in detail herein, the PDZ proteins listed in Table 2 are native PDZ domain-containing proteins. Only important interactions are shown in this table. Therefore, the present invention is particularly directed to the detection and regulation of the interaction between PDZ protein and PL protein. PDZ domains that bind other pathogens can be used in a similar fashion for the diagnosis of infection. Additional examples of pathogenic PL proteins suitable for diagnosis are included in Table 8, although this is not meant to limit the scope of the invention.

[0099] In another embodiment of the present invention, cell abnormalities or diseases can be diagnosed by detecting im...

Embodiment 1

[0357] Sequence analysis of HPV E6 protein to determine oncogenic potential

[0358] PDZ proteins are known to bind to the carboxy-terminal sequence (PL) of certain proteins. The PL sequences that bind PDZ domains are predictable and are described in detail in US Patent Applications 09 / 710059, 09 / 724553 and 09 / 688017. A group of proteins ending with the sequence -X-(S / T)-X-(V / I / L) is one of the important classes of PL motifs. We have examined the C-terminal sequence of the E6 protein from several HPV strains. All oncogenic virus strains identified by the National Cancer Institute share a common PDZ-binding sequence. Those non-oncogenic E6 proteins from papillomavirus strains were not predicted to bind PDZ domain sequences, thus implying that the interaction with PDZ proteins is a prerequisite for their cancer-causing in humans. In the sequences examined (Table 3A), there was a 100% correlation between the presence of PL and the initiation of cancer. In theory, using the PL...

Embodiment 2

[0368] Identification of PDZ domains that interact with the C-terminus of oncogenic E6 proteins

[0369] To identify PDZ domains that can be used to detect oncogenic E6 proteins in diagnostic assays, the 'G test' (described above) was used to identify interactions between E6PL and PDZ domains. Peptides were synthesized according to the C-terminal amino acid sequence of the E6 protein from an oncovirus strain of human papillomavirus. The ability of these peptides to bind the PDZ domain was assessed using the G assay described above and the PDZ domain synthesized from the expression constructs detailed in US Patent Applications 09 / 710059, 09 / 724553 and 09 / 688017. The results of these assays showing high binding affinities are listed in Table 4 below.

[0370] As we will see below, there are a large number of PDZ domains that bind some oncogenic E6 proteins. However, it appears that only the second PDZ domain of MAGI-1 binds all tested oncogenic E6PLs. The PDZ domain of TIP-1 ...

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Abstract

The invention provides reagents and methods for detecting pathogen infections in human samples. This detection utilizes specific proteins to detect the presence of pathogen proteins or abnormal expression of human proteins resulting from pathogen infections. Specific methods, compositions and kits are disclosed herein for the detection of oncogenic human papillomavirus E6 proteins in clinical samples.

Description

[0001] cross reference [0002] This application claims U.S. Patent Application No. 10 / 630,590 filed July 29, 2003, U.S. Provisional Application No. 60 / 490,094 filed July 25, 2003, U.S. Provisional Application No. 60 / 450,464 and U.S. Provisional Application No. 60 / 409,298, filed September 9, 2002. field of invention [0003] The present invention relates to the detection of biological markers derived from pathogenic organisms, such as observed in certain human papillomavirus (HPV) infections, and the use of such diagnostics to identify infections that may lead to cancer or other disorders of samples. Said invention also discloses a composition, method and kit for detecting oncogenic HPV E6 protein in clinical samples as a means of tumor diagnosis. Background of the invention [0004] Cervical cancer is the second most common neoplasm in women and is currently associated with 99.7% of high-risk HPV infections. Now, 12,000 new cases of invasive cervical cancer are diagnose...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53C12N15/09G01N33/543G01N33/569G01N33/574
CPCG01N33/57411G01N33/56983
Inventor P·S·鲁J·施韦策C·S·迪亚斯-萨米恩托M·P·贝尔马斯
Owner 生命树股份有限公司
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