PubMed · 62238
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L Spence, M Fauvel, R Petro, S Bloch. 1976-11-06. Haemagglutinin from Rotavirus.. https://doi.org/10.1016/s0140-6736(76)90860-6
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A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.
The lymphocytosis promoting factor-haemagglutinin of Bordetella pertussis was isolated from solutions obtained after cell disintegration by a novel affinity chromatographic method using an adsorbent composed of human haptoglobin covalently attached to a Sepharose 4B matrix. The haemagglutinin was bound to the adsorbent at pH 6.5 and eluted by a stepwise change to a pH 10 buffer. A 300--600-fold purification of the haemagglutinin was achieved by this one-step process. The chemical and biological properties of the haemagglutinin isolated by affinity chromatography were found to be similar to those of the protein isolated by other workers from culture supernatants. The affinity chromatographic method was found to be specific for the purification of the lymphocytosis promoting factor-haemagglutinin and no purification of the fimbrial-haemagglutinin of Bordetella pertussis was achieved by the method.