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

An alu sequence and vaccine technology, applied in the field of Alu sequence of African green monkeys, can solve problems such as undeveloped probes, and achieve the effect of monitoring residual DNA content

Active Publication Date: 2008-09-24
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] Using the African green monkey Alu sequence X01476 in GeneBank as a reference design PCR primers. 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, SEQID NO: 4). The PCR reaction parameters are: denaturation at 98°C for 5 minutes, 25sec at 94°C, 30sec at 68°C, 30sec at 72°C, 30 cycles, and extension at 72°C for 10 minutes. After PCR amplification, a 290bp band ( figure 1 ).

[0024] (2) Cloning of Alu sequence

[0025] The PCR product was cloned on Promega's pGEM-T and transformed into E.coli JM109 strain. After a small amount of plasmid was extracted, NcoI and SalI were identified by double enzyme digestion, and several positive clones containing 290bp insert were obtained ( figure 2 ), select a clone named T-Alu, its structure is as image 3 Shown.

Embodiment 2

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

[0027] 300ng purified and recovered Alu sequence NcoI and SalI digested fragments were labeled according to the instructions of Roche's Digoxin DNA Labeling and Detection Kit. The detection of residual DNA in SARS vaccine adopts dot blot method.

[0028] First, cut a piece of nylon membrane of the required size, respectively spot the Ver0DNA reference standards of 10gn, 1ng, 100pg, and 10pg on the membrane, and then spot the sample to be tested on the membrane according to different dilutions. The membranes were respectively subjected to denaturation solution (0.5M NaOH, 1.5M NaCl), neutralization solution (0.5M Tris pH7.0, 1M NaCl), 2XSSC for 5 minutes each, and then crosslinked in a UV crosslinker at 45J for 5 minutes. The membrane was pre-hybridized in the hybridization bag for 1 hour, and then probes were added for hybridization overnight. After washing the membrane, the ...

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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

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

Claims

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Application Information

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Patent Type & Authority Patents(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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