Molecular Cloning Of A CDNA Encoding A Specific Antigenic Polypeptide Of Cysticercus Cellulosae

Objective: Cysticercosis is a kind of Amphixenosis caused by cysticercus ,and has a wide distribution in the world . Cysticercus is a kind of parasite which often lives in many organs such as brain, eyes ,heart and so on . When living in nervous system ,it can cause serious disease ,for example cerebral cysticercosis which does great harm to health of human. However, the current diagnosis of cysticercosis still makes the results of CT or MR as main standard. Due to the high cost of CT or MR, it is impossible to evaluate curative effect and carry out mass epidemic survey . The key to solve these problem lies in seeking a convenient ,efficient and accurate method of diagnosis. With the development in the technology of genetic engineering , it is possible to obtain antigens with high specificity and sensibitity in diagnosis. In this study the cDNA expression library of cysticercosis was immunoscreened by the patient serum of cerebral cysticercosis in order to clone a gene encoding antigen used in specific diagnosis and prevention of cysticercosis.Methods : STEP ONE: The obtaining of serum absorbed by the splitting solution of E. coli: Dilute the serum of cerebral cysticercosis in TBS buffer. Mix fully the splitting solution ofXL1-Blue MRF' with the diluted serum, room temperature 4 h. After centrifugation, the serum absorbed by the splitting solution of E.coli was obtained. STEP TWO: The immunoscreening of cDNA expression library of cysticercus cellulosae: Using above absorbed serum as first -antibody and Anti-human IgG HRP as second -antibody, the cDNA library was screened. One positive phage containing cDNA was isolated. After the cultivation and amplification of the positive phage , the phage DNA was extracted. STEP THREE: The PCR amplification of the inserted positive cDNA: Using the templats of the extracted positive phage DNA and universal primers ( T3 and T7 ) located at the two ends of the inserted cDNA, PCR was performed under the following condition: 94℃ for 5 min, then 94℃ for 1 min ,56℃ for 1 min and 72℃ for 1 min per cycle for 30 cycles , last 72℃ for 10 min . STEP FOUR: The purified PCR product was digested by two kinds of Restriction Endonucleases ( EcoR I and Kpn I), which obtain two different cohesive ends .Then the digested segment was ligated into PUC18 plasmid vector and transformed into the competent E.coli JM 109. According to the principle of α-complement of β-galactosidase , select the white colony on the LB culture with ampicillin ,IPTG and x-gal. The positive clones were identified by enzyme digestion . STEP FIVE: The sequencing and homologic analysis of the cDNA. The nucleotide sequence of the cDNA was determinated by Sanger dideoxynucleotide chaintermination method. The amino acid sequence was deduced from nucleotide sequence using GENETYX software. The homological search of the nucleotide and amino acid sequence was done by using BLASTN and BLASTP in NCBI. STEP SIX:Northern blotting. Northern blotting was done in order to measure the size of the mRNA in cysticercus cellulosae : Firstly total RNA was extracted from the cysticercus cellulosae by the single step of acid guanidium thiocynaate-phenol-chloroform. Identify the integrity ,purity and content of mRNA by electrophoresis and ultraviolet spectrophotometer . Secondly Electrophorese mRNA in the agarose gel denatured by Formaldehyd and transfer them into Nylon membrane by siphon . Using the positive clone as templat , label the probe with isotope. Next the RNAs on the blot were hybridized to the labeled probe for the positive clone , and the blot was then exposed to x-ray film .Results : A cDNA clone of 553 bp, named cYI, was isolated. The result of Northern blotting has shown that there was a band of 0.6 kb . The clone contained one opening reading frame (between 2 and 484 bp) of 553 bp encoding 160 amino acid residues (MV=18.34kDa). The initiation codon of 5' end was not found . The termination codon (TAA), poly (A) single (AATAAA) and poly(A) tail were all found in 3' end. The compose of nucleotid...