Alu sequence of Africa green monkey for detecting content of residual DNA in SARS vaccine

A kind of alu sequence and content technology, used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve problems such as undeveloped probes, and achieve the effect of monitoring residual DNA content

Active Publication Date: 2005-11-02
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] A simple, reproducible and stable probe has not been developed for the detection of residual DNA in vaccines prepared on Vero cells

Method used

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  • Alu sequence of Africa green monkey for detecting content of residual DNA in SARS vaccine
  • Alu sequence of Africa green monkey for detecting content of residual DNA in SARS vaccine
  • Alu sequence of Africa green monkey for detecting content of residual DNA in SARS vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of African green monkey Alu sequence

[0022] PCR method to amplify Alu sequence

[0023] PCR primers were designed using the African green monkey Alu sequence X01476 in GeneBank as a reference. Upstream primer P1: 5'-gct, ttg, agg, ccg, ggc, ggg, atg-3' (21bp, SEQ ID NO: 3), downstream primer P2: 5'-ctc, tct, ctt, aga, gtc, ttg , ctc, tg-3' (23bp, SEQ ID NO: 4). The PCR reaction parameters were: denaturation at 98°C for 5 min, 30 cycles at 94°C for 25 sec, 30 sec at 68°C, and 30 sec at 72°C, and extension at 72°C for 10 min. After PCR amplification, a 290bp band ( figure 1 ).

[0024] (2) Cloning of Alu sequence

[0025] The PCR product was cloned on the pGEM-T of Promega Company, transformed into E.coliJM109 bacterial strain, and by a small amount of plasmid extraction, NcoI and SalI double enzyme digestion identification, obtained several positive clones containing 290bp inserts ( figure 2 ), select a clone named T-Alu, its structure is a...

Embodiment 2

[0026] Example 2 Method for detecting residual DNA in vaccines using African green monkey Alu sequence

[0027] 300 ng of purified and recovered Alu sequence NcoI and SalI fragments were labeled according to the instructions of Roche Digoxigenin DNA Labeling and Detection Kit. The detection of residual DNA in the SARS vaccine was done by dot blot.

[0028] First, cut out a piece of nylon membrane of the required size, respectively spot 10gn, 1ng, 100pg, and 10pg Vero DNA reference standards on the membrane, and then spot the samples to be tested on the membrane according to different dilutions. The membrane was passed through denaturing solution (0.5M NaOH, 1.5M NaCl), neutralizing solution (0.5M Tris pH7.0, 1M NaCl), 2XSSC for 5 minutes each, and then cross-linked at 45J for 5 minutes in an ultraviolet crosslinker. Pre-hybridize the membrane in the hybridization bag for 1 hour, then add the probe to hybridize overnight, wash the membrane, add anti-digoxigenin secondary antib...

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Abstract

The invention relates to the African green monkey Alu sequence using to detect the residual content of DNA of the vaccine against SARS, and to the detection method with the related sequence.

Description

field of invention [0001] The invention relates to the Alu sequence of the African green monkey used for detecting the residual DNA content of the SARS vaccine, and a detection method using the sequence. Background of the invention [0002] The Alu sequence is a moderately repetitive sequence on the chromosomes of humans, primates and rodents. It is named because it contains a restriction endonuclease Alu site, with about 300,000 copies. The Alu sequence is about 280bp long, the left half of the sequence 120bp and the right half 150bp are two similar units, separated by an A-rich region in the middle, the 5' end is relatively conserved in different Alu families, and the 3' end is composed of Compare the varied poly A composition. There are 7-20bp direct repeats flanking the Alu sequence, which are not conserved in different Alu families, but unique (Schmid CW, Jelinek WR. The Alu family of dispersed repetitive sequences. Science, 1982, 216: 1065-1070) . Grimaldi et al. st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 高虹张振山高强韩中山宁叶刘长民张建三尹卫东
Owner SINOVAC BIOTECH
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