Specific antibody against ubiquitin ligase nedd4‑1 and its application
A ubiquitin ligase, specific technology, applied in the direction of ligase, anti-enzyme immunoglobulin, enzyme, etc.
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Embodiment 1
[0026] Example 1: Design and expression of Nedd4-1 antigen Ag5 and production of its antibody.
[0027] 1. Specific antigen selection of anti-Nedd4-1 antibody. Nedd4-1 and Nedd4-2 in the Nedd4 ubiquitin ligase family have a high degree of similarity in their amino acid sequences, and about 70% of the sequences are identical. Existing anti-Nedd4-1 antibodies can also react with Nedd4-2. In order to design a highly specific anti-Nedd4-1 antibody, we analyzed the sequences of Nedd4-1 and Nedd4-2, and selected a sequence P224-F282 with the lowest similarity between Nedd4-1 and Nedd4-2: PQDNLTDAENGNIQLQAQRAFTTRR QISEETESVDNQESSENWEIIREDEATMYSSQAF(SEQ ID NO.1) (see figure 1 ), we named this specific antigen of Nedd4-1 Ag5.
[0028] 2. Expression and purification of Nedd4-1 antigen Ag5:
[0029]The cDNA of Ag5 was cloned into the E. coli expression vector pGEX4T3 of GST fusion protein (GE Healthcare Life Sciences, Piscataway, NJ, USA) to obtain the plasmid pGEX4T3-GST-Ag5. The s...
Embodiment 2
[0039] Example 2: The expression of Nedd4-1 in lung cancer tissues was detected using an anti-Nedd4-1Ag5 antibody kit.
[0040] (i) Lung cancer tissue samples. 71 cases of lung cancer tissue and adjacent normal tissue samples were obtained from the lung cancer tissue sample bank of the Oncology Department of Shanghai Changzheng Hospital.
[0041] (ii) Immunohistochemical staining step. We performed immunohistochemical staining on microarray tissue samples of lung cancer and adjacent normal tissues with anti-Ag5 antibody kit and anti-EGFR antibody to detect the expression of Nedd4-1 and EGFR in 71 lung cancer samples. The specific steps are briefly described as follows: firstly, the paraffin-embedded 4 μm sliced tissue microarray was deparaffinized and rehydrated with xylene and alcohol solution. For antigen retrieval, the sections were treated with 0.01M citrate buffer (pH 6.0) at 98°C for 5 minutes (microwave treatment), and then the sections were cooled to room temperatu...
Embodiment 3
[0044] Example 3: The expression of Nedd4-1 in cardia cancer tissue was detected by using an anti-Nedd4-1Ag5 antibody kit.
[0045] (i) Cardia cancer tissue samples. 214 gastric cancer tissue samples were obtained from the Gastric Cancer Tissue Bank of the Oncology Department of Shanghai Changzheng Hospital. The clinical data of cardiac cancer samples are shown in Table 1. Age distribution of cardiac cancer patients: 106 people over 60 years old, 108 people 60 or under 60 years old. There were 157 males and 57 females. Pathological indicators: 48 cases of tumors above 6 cm, 166 cases of tumors of 6 cm or less; 130 cases of tumors with high or moderate degree of differentiation, 84 cases of tumors with low or undifferentiated degree; 63 cases of tumors with invasion degree I / II, grade III / IV 151 cases; 73 cases without lymph node metastasis, 141 cases with metastasis; 83 cases of TNM I / II grade, 131 cases of III / IV grade. These clinical data showed that malignant cardia car...
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